Isolation of endothelial cells, pericytes and astrocytes from mouse brain


Autoři: Florian Bernard-Patrzynski aff001;  Marc-André Lécuyer aff002;  Ina Puscas aff001;  Imane Boukhatem aff001;  Marc Charabati aff002;  Lyne Bourbonnière aff002;  Charles Ramassamy aff004;  Grégoire Leclair aff001;  Alexandre Prat aff002;  V Gaëlle Roullin aff001
Působiště autorů: Faculty of Pharmacy, Université de Montréal, Montreal, Québec, Canada aff001;  Department of Neuroscience, Faculty of Medicine, Université de Montréal, Montreal, Québec, Canada aff002;  Institute for Multiple Sclerosis Research and Neuroimmunology, University Medical Center Göttingen, Göttingen, Germany aff003;  Institut National de la Recherche Scientifique, Armand-Frappier Institute, Laval, Québec, Canada aff004
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226302

Souhrn

Primary cell isolation from the central nervous system (CNS) has allowed fundamental understanding of blood-brain barrier (BBB) properties. However, poorly described isolation techniques or suboptimal cellular purity has been a weak point of some published scientific articles. Here, we describe in detail how to isolate and enrich, using a common approach, endothelial cells (ECs) from adult mouse brains, as well as pericytes (PCs) and astrocytes (ACs) from newborn mouse brains. Our approach allowed the isolation of these three brain cell types with purities of around 90%. Furthermore, using our protocols, around 3 times more PCs and 2 times more ACs could be grown in culture, as compared to previously published protocols. The cells were identified and characterized using flow cytometry and confocal microscopy. The ability of ECs to form a tight monolayer was assessed for passages 0 to 3. The expression of claudin-5, occludin, zonula occludens-1, P-glycoprotein-1 and breast cancer resistance protein by ECs, as well as the ability of the cells to respond to cytokine stimuli (TNF-α, IFN-γ) was also investigated by q-PCR. The transcellular permeability of ECs was evaluated in the presence of pericytes or astrocytes in a Transwell® model by measuring the transendothelial electrical resistance (TEER), dextran-FITC and sodium fluorescein permeability. Overall, ECs at passages 0 and 1 featured the best properties valued in a BBB model. Furthermore, pericytes did not increase tightness of EC monolayers, whereas astrocytes did regardless of their seeding location. Finally, ECs resuspended in fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) could be cryopreserved in liquid nitrogen without affecting their phenotype nor their capacity to form a tight monolayer, thus allowing these primary cells to be used for various longitudinal in vitro studies of the blood-brain barrier.

Klíčová slova:

Astrocytes – Confocal microscopy – Endothelial cells – Flow cytometry – Permeability – Primary cells – Pericytes – Electrical resistance


Zdroje

1. Banerjee J, Shi Y, Azevedo HS. In vitro blood–brain barrier models for drug research: state-of-the-art and new perspectives on reconstituting these models on artificial basement membrane platforms. Drug Discovery Today. 2016;21(9):1367–86. doi: 10.1016/j.drudis.2016.05.020 27283274

2. Stamatovic SM, Johnson AM, Keep RF, Andjelkovic AV. Junctional proteins of the blood-brain barrier: New insights into function and dysfunction. Tissue Barriers. 2016;4(1):e1154641. doi: 10.1080/21688370.2016.1154641 PMC4836471. 27141427

3. Engelhardt B, Sorokin L. The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction. Semin Immunopathol. 2009;31(4):497–511. doi: 10.1007/s00281-009-0177-0 19779720; PubMed Central PMCID: PMC19779720.

4. Lécuyer M-A, Kebir H, Prat A. Glial influences on BBB functions and molecular players in immune cell trafficking. Biochimica et Biophysica Acta (BBA)—Molecular Basis of Disease. 2016;1862(3):472–82. doi: https://doi.org/10.1016/j.bbadis.2015.10.004

5. Pardridge WM. The blood-brain barrier: Bottleneck in brain drug development. NeuroRX. 2005;2(1):3–14. doi: 10.1602/neurorx.2.1.3 15717053

6. Sharif Y, Jumah F, Coplan L, Krosser A, Sharif K, Tubbs RS. Blood brain barrier: A review of its anatomy and physiology in health and disease. Clinical anatomy (New York, NY). 2018;31(6):812–23. Epub 2018/04/11. doi: 10.1002/ca.23083 29637627.

7. Modarres HP, Janmaleki M, Novin M, Saliba J, El-Hajj F, RezayatiCharan M, et al. In vitro models and systems for evaluating the dynamics of drug delivery to the healthy and diseased brain. Journal of Controlled Release. 2018;273:108–30. doi: 10.1016/j.jconrel.2018.01.024 29378233

8. Lorsch JR, Collins FS, Lippincott-Schwartz J. Cell Biology. Fixing problems with cell lines. Science (New York, NY). 2014;346(6216):1452–3. doi: 10.1126/science.1259110 25525228.

9. Development Organization Workgroup Asn ATCCS. Cell line misidentification: the beginning of the end. Nature Reviews Cancer. 2010;10:441. doi: 10.1038/nrc2852 https://www.nature.com/articles/nrc2852#supplementary-information. 20448633

10. Czupalla CJ, Liebner S, Devraj K. In Vitro Models of the Blood–Brain Barrier. In: Milner R, editor. Cerebral Angiogenesis: Methods and Protocols. New York, NY: Springer New York; 2014. p. 415–37.

11. Larochelle C, Cayrol R, Kebir H, Alvarez JI, Lécuyer M-A, Ifergan I, et al. Melanoma cell adhesion molecule identifies encephalitogenic T lymphocytes and promotes their recruitment to the central nervous system. Brain. 2012;135(Pt 10):2906–24. doi: 10.1093/brain/aws212 22975388; PubMed Central PMCID: PMC22975388.

12. Lécuyer M-A, Saint-Laurent O, Bourbonnière L, Larouche S, Larochelle C, Michel L, et al. Dual role of ALCAM in neuroinflammation and blood–brain barrier homeostasis. Proceedings of the National Academy of Sciences. 2017;114(4):E524–E33. doi: 10.1073/pnas.1614336114 28069965

13. Deli M, Ábrahám C, Kataoka Y, Niwa M. Permeability Studies on In Vitro Blood–Brain Barrier Models: Physiology, Pathology, and Pharmacology. Cell Mol Neurobiol. 2005;25(1):59–127. doi: 10.1007/s10571-004-1377-8 15962509

14. Justice MJ, Dhillon P. Using the mouse to model human disease: increasing validity and reproducibility. Disease Models & Mechanisms. 2016;9(2):101–3. doi: 10.1242/dmm.024547 26839397

15. Chinwalla AT, Cook LL, Delehaunty KD, Fewell GA, Fulton LA, Fulton RS, et al. Initial sequencing and comparative analysis of the mouse genome. Nature. 2002;420(6915):520–62. doi: 10.1038/nature01262 12466850

16. Goto F, Goto K, Weindel K, Folkman J. Synergistic effects of vascular endothelial growth factor and basic fibroblast growth factor on the proliferation and cord formation of bovine capillary endothelial cells within collagen gels. Laboratory investigation; a journal of technical methods and pathology. 1993;69(5):508–17. 8246443.

17. Woost PG, Jumblatt MM, Eiferman RA, Schultz GS. Growth factors and corneal endothelial cells: I. Stimulation of bovine corneal endothelial cell DNA synthesis by defined growth factors. Cornea. 1992;11(1):1–10. doi: 10.1097/00003226-199201000-00001 1559341.

18. Calabria AR, Weidenfeller C, Jones AR, De Vries HE, Shusta EV. Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction. Journal of Neurochemistry. 2006;97(4):922–33. doi: 10.1111/j.1471-4159.2006.03793.x 16573646

19. Perrière N, Demeuse P, Garcia E, Regina A, Debray M, Andreux J-P, et al. Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood–brain barrier-specific properties. Journal of Neurochemistry. 2005;93(2):279–89. doi: 10.1111/j.1471-4159.2004.03020.x 15816851

20. Cayrol R, Wosik K, Berard JL, Dodelet-Devillers A, Ifergan I, Kebir H, et al. Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system. Nat Immunol. 2008;9(2):137–45. doi: 10.1038/ni1551 18157132; PubMed Central PMCID: PMC18157132.

21. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic acids research. 2001;29(9):e45–e. doi: 10.1093/nar/29.9.e45 11328886.

22. Avdeef A. How well can in vitro brain microcapillary endothelial cell models predict rodent in vivo blood-brain barrier permeability? European Journal of Pharmaceutical Sciences. 2011;43(3):109–24. doi: 10.1016/j.ejps.2011.04.001 WOS:000291906500003. 21514381

23. Kumar A, D’Souza SS, Moskvin OV, Toh H, Wang B, Zhang J, et al. Specification and Diversification of Pericytes and Smooth Muscle Cells from Mesenchymoangioblasts. Cell Reports. 2017;19(9):1902–16. doi: 10.1016/j.celrep.2017.05.019 28564607

24. Saraswati S, Marrow SMW, Watch LA, Young PP. Identification of a pro-angiogenic functional role for FSP1-positive fibroblast subtype in wound healing. Nature Communications. 2019;10(1):3027. doi: 10.1038/s41467-019-10965-9 31289275

25. Middeldorp J, Hol EM. GFAP in health and disease. Progress in Neurobiology. 2011;93(3):421–43. doi: 10.1016/j.pneurobio.2011.01.005 21219963

26. Raponi E, Agenes F, Delphin C, Assard N, Baudier J, Legraverend C, et al. S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage. Glia. 2007;55(2):165–77. doi: 10.1002/glia.20445 17078026.

27. Abbott NJ, Rönnbäck L, Hansson E. Astrocyte–endothelial interactions at the blood–brain barrier. Nature Reviews Neuroscience. 2006;7:41. doi: 10.1038/nrn1824 16371949

28. Xu Y, Xie X, Duan Y, Wang L, Cheng Z, Cheng J. A review of impedance measurements of whole cells. Biosensors and Bioelectronics. 2016;77:824–36. doi: 10.1016/j.bios.2015.10.027 26513290

29. Kangwantas K, Pinteaux E, Penny J. The extracellular matrix protein laminin-10 promotes blood-brain barrier repair after hypoxia and inflammation in vitro. J Neuroinflammation. 2016;13:25. Epub 2016/02/03. doi: 10.1186/s12974-016-0495-9 26832174; PubMed Central PMCID: PMC4736307.

30. Bhargavan B, Kanmogne GD. Differential Mechanisms of Inflammation and Endothelial Dysfunction by HIV-1 Subtype-B and Recombinant CRF02_AG Tat Proteins on Human Brain Microvascular Endothelial Cells: Implications for Viral Neuropathogenesis. Mol Neurobiol. 2018;55(2):1352–63. Epub 2017/01/28. doi: 10.1007/s12035-017-0382-0 28127697.

31. Banks WA, Gray AM, Erickson MA, Salameh TS, Damodarasamy M, Sheibani N, et al. Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit. J Neuroinflammation. 2015;12:223. Epub 2015/11/27. doi: 10.1186/s12974-015-0434-1 26608623; PubMed Central PMCID: PMC4660627.

32. Rochfort KD, Collins LE, McLoughlin A, Cummins PM. Tumour necrosis factor‐α‐mediated disruption of cerebrovascular endothelial barrier integrity in vitro involves the production of proinflammatory interleukin‐6. Journal of Neurochemistry. 2016;136(3):564–72. doi: 10.1111/jnc.13408 26499872

33. Cornford EM, Hyman S. Localization of brain endothelial luminal and abluminal transporters with immunogold electron microscopy. NeuroRx: the journal of the American Society for Experimental NeuroTherapeutics. 2005;2(1):27–43. Epub 2005/02/18. doi: 10.1602/neurorx.2.1.27 15717055; PubMed Central PMCID: PMC539318.

34. Bicker J, Alves G, Fortuna A, Falcão A. Blood–brain barrier models and their relevance for a successful development of CNS drug delivery systems: A review. European Journal of Pharmaceutics and Biopharmaceutics. 2014;87(3):409–32. doi: 10.1016/j.ejpb.2014.03.012 24686194

35. Choudhury AR. A comprehensive review of cell isolation methods. MATER METHODS. 2017;7(2260). doi: 10.13070/mm.en.7.2260

36. Souza DG, Bellaver B, Souza DO, Quincozes-Santos A. Characterization of Adult Rat Astrocyte Cultures. PLoS ONE. 2013;8(3):e60282. doi: 10.1371/journal.pone.0060282 23555943

37. Boroujerdi A, Tigges U, Welser-Alves J, Milner R. Isolation and Culture of Primary Pericytes from Mouse Brain. In: Milner R, editor. Cerebral Angiogenesis. Methods in Molecular Biology. 1135: Springer New York; 2014. p. 383–92.

38. Tigges U, Welser-Alves JV, Boroujerdi A, Milner R. A novel and simple method for culturing pericytes from mouse brain. Microvascular Research. 2012;84(1):74–80. doi: 10.1016/j.mvr.2012.03.008 22484453

39. de Boer PJG A. G. The Blood–Brain Barrier and its Effect on Absorption and Distribution. Preclinical Development Handbook.

40. Sun X, Hu X, Wang D, Yuan Y, Qin S, Tan Z, et al. Establishment and characterization of primary astrocyte culture from adult mouse brain. Brain Research Bulletin. 2017;132:10–9. doi: 10.1016/j.brainresbull.2017.05.002 28499803

41. Takata F, Dohgu S, Yamauchi A, Matsumoto J, Machida T, Fujishita K, et al. In Vitro Blood-Brain Barrier Models Using Brain Capillary Endothelial Cells Isolated from Neonatal and Adult Rats Retain Age-Related Barrier Properties. PLOS ONE. 2013;8(1):e55166. doi: 10.1371/journal.pone.0055166 23383092

42. Chen J, Luo Y, Hui H, Cai T, Huang H, Yang F, et al. CD146 coordinates brain endothelial cell-pericyte communication for blood-brain barrier development. Proc Natl Acad Sci U S A. 2017;114(36):E7622–E31. Epub 2017/08/21. doi: 10.1073/pnas.1710848114 28827364.

43. Assmann JC, Müller K, Wenzel J, Walther T, Brands J, Thornton P, et al. Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice. Bio-protocol. 2017;7(10):e2294. doi: 10.21769/BioProtoc.2294 28603749.

44. Welser-Alves JV, Boroujerdi A, Milner R. Isolation and Culture of Primary Mouse Brain Endothelial Cells. In: Milner R, editor. Cerebral Angiogenesis: Methods and Protocols. New York, NY: Springer New York; 2014. p. 345–56.

45. Ruck T, Bittner S, Epping L, Herrmann AM, Meuth SG. Isolation of primary murine brain microvascular endothelial cells. Journal of visualized experiments: JoVE. 2014;(93):e52204–e. doi: 10.3791/52204 25489873.

46. Wylot B, Konarzewska K, Bugajski L, Piwocka K, Zawadzka M. Isolation of vascular endothelial cells from intact and injured murine brain cortex—technical issues and pitfalls in FACS analysis of the nervous tissue. Cytometry Part A. 2015;87(10):908–20. doi: 10.1002/cyto.a.22677 25892199

47. Yousef H, Czupalla CJ, Lee D, Butcher EC, Wyss-Coray T. Papain-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry. Bio-protocol. 2018;8(22):e3091. doi: 10.21769/BioProtoc.3091 31032379.

48. Lochhead JJ, Ronaldson PT, Davis TP. Hypoxic Stress and Inflammatory Pain Disrupt Blood-Brain Barrier Tight Junctions: Implications for Drug Delivery to the Central Nervous System. The AAPS Journal. 2017;19(4):910–20. doi: 10.1208/s12248-017-0076-6 28353217

49. Banks WA, Gray AM, Erickson MA, Salameh TS, Damodarasamy M, Sheibani N, et al. Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit. Journal of Neuroinflammation. 2015;12(1):223. doi: 10.1186/s12974-015-0434-1 26608623

50. Wolff A, Antfolk M, Brodin B, Tenje M. In Vitro Blood-Brain Barrier Models-An Overview of Established Models and New Microfluidic Approaches. J Pharm Sci. 2015. doi: 10.1002/jps.24329 25630899; PubMed Central PMCID: PMC25630899.

51. Feldmann M, Pathipati P, Sheldon RA, Jiang X, Ferriero DM. Isolating astrocytes and neurons sequentially from postnatal murine brains with a magnetic cell separation technique. Journal of Biological Methods. 2014;1(2):e11.

52. Schildge S, Bohrer C, Beck K, Schachtrup C. Isolation and Culture of Mouse Cortical Astrocytes. 2013;(71):e50079. doi: 10.3791/50079 23380713

53. Saura J. Microglial cells in astroglial cultures: a cautionary note. Journal of Neuroinflammation. 2007;4(1):26. doi: 10.1186/1742-2094-4-26 17937799

54. Nakagawa S, Deli MA, Kawaguchi H, Shimizudani T, Shimono T, Kittel Á, et al. A new blood–brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochemistry International. 2009;54(3–4):253–63. doi: 10.1016/j.neuint.2008.12.002 19111869

55. Neng L, Zhang W, Hassan A, Zemla M, Kachelmeier A, Fridberger A, et al. Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the young mouse ear. Nat Protocols. 2013;8(4):709–20. doi: 10.1038/nprot.2013.033 23493068

56. Crouch EE, Doetsch F. FACS isolation of endothelial cells and pericytes from mouse brain microregions. Nature Protocols. 2018;13:738. doi: 10.1038/nprot.2017.158 https://www.nature.com/articles/nprot.2017.158#supplementary-information. 29565899

57. Yao Y, Chen Z-L, Norris EH, Strickland S. Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity. Nature communications. 2014;5:3413–. doi: 10.1038/ncomms4413 PMC3992931. 24583950

58. Dore-Duffy P, Esen N. The Microvascular Pericyte: Approaches to Isolation, Characterization, and Cultivation. In: Birbrair A, editor. Pericyte Biology—Novel Concepts. Cham: Springer International Publishing; 2018. p. 53–65.


Článek vyšel v časopise

PLOS One


2019 Číslo 12