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BRAIN Publication-derived

Lyme Neuroborreliosis Brain Organoids

Full SOP with Academic or a plan

Source Adams et al., 2023 · Centre for Medical Parasitology, Department of Immunology and Microbiology, University of Copenhagen · 10.1016/j.isci.2022.105838

Authors: Yvonne Adams, Anne Skovsbo Clausen, Peter Østrup Jensen, Malin Lager, Peter Wilhelmsson, Anna J. Henningson, Per-Eric Lindgren, Daniel Faurholt-Jepsen, Helene Mens, Peter Kraiczy, Kasper Nørskov Kragh, Thomas Bjarnsholt, Andreas Kjaer, Anne-Mette Lebech, Anja R. Jensen Duration: 9 days 6 phases Cell source: Human iPSC-derived (hCMEC/D3 endothelial cells, primary astrocytes, pericytes)

Abstract

This protocol describes the generation and use of 3D blood-brain barrier (BBB) organoids composed of human brain endothelial cells, pericytes, and astrocytes to model Lyme neuroborreliosis. The organoids are used to assess the ability of Borrelia burgdorferi genospecies to invade the BBB, induce structural changes, and disrupt tight junctions, providing a human-relevant in vitro model for studying spirochetal CNS infection.

Cell source
Human iPSC-derived (hCMEC/D3 endothelial cells, primary astrocytes, pericytes)
Application
Disease modeling

Protocol overview

25 steps across 6 phases

Phase 1 · BBB-Organoid Generation and Assembly Days 1-3
  1. a Prepare agarose-coated wells
  2. b Prepare cell suspension for organoid seeding
  3. c Seed multicellular organoids
  4. d Allow organoid self-assembly
  5. e Verify cellular orientation by astrocyte staining
Phase 2 · Borrelia Preparation and Co-culture Days 7-8
  1. a Culture Borrelia spirochetes
  2. b Stain spirochetes for flow cytometry
  3. c Wash and resuspend spirochetes
  4. d Final spirochete preparation for co-culture
  5. e Co-incubate Borrelia with BBB-organoids
Phase 3 · Fixation, Staining, and Confocal Imaging Days 8-9
  1. a Pool and fix organoids
  2. b Prepare organoids for imaging
  3. c Confocal microscopy imaging
  4. d Quantify spirochete invasion by volume
Phase 4 · Organoid Morphometric Analysis Days 8-9
  1. a Calculate organoid volume
  2. b Calculate organoid roundness
  3. c Calculate organoid circularity
Phase 5 · Tight Junction Protein Analysis Days 8-9
  1. a Fix organoids and permeabilize
  2. b Block non-specific binding and stain ZO-1
  3. c Apply secondary antibody
  4. d Stain nuclei and image
  5. e Quantify tight junction disruption
Phase 6 · Live/Dead Cell Viability Assessment Days 8-9
  1. a Prepare organoids and positive control
  2. b Apply Live/Dead stain
  3. c Image and quantify cell death

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Attribution

This SOP was written by Organthis based on the published method in Adams et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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