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BRAIN Publication-derived

Cryptococcal Meningitis Cerebral Organoids

Full SOP with Academic or a plan

Source Harding et al., 2025 · Massachusetts General Hospital, Harvard Medical School · 10.20944/preprints202506.2186.v1

Authors: Alfred T. Harding, Lee Gehrke, Jatin M. Vyas, Hannah Brown Harding Duration: 35 days 8 phases Cell source: Human ESC (WIBR3)

Abstract

This protocol describes the generation of human cerebral organoids from embryonic stem cells and their use as a model for studying Cryptococcus neoformans infection and dissemination. The organoids accurately recapitulate brain cell types and structures, allowing investigation of fungal pathogenesis, tissue penetration, and host immune responses including cytokine release and inflammatory transcriptional changes.

Cell source
Human ESC (WIBR3)
Application
Disease modeling

Protocol overview

69 steps across 8 phases

Phase 1 · Organoid Generation and Maturation 0-35 days
  1. a Embryoid body formation
  2. b Neural induction
  3. c Matrigel embedding
  4. d Orbital shaker culture
  5. e Organoid maturation checkpoint
Phase 2 · Fungal Infection of Organoids Day 0-21 post-infection
  1. a Fungal culture preparation
  2. b Fungal cell harvesting and counting
  3. c Organoid infection inoculation
  4. d Infected organoid culture maintenance
  5. e Uninfected control maintenance
Phase 3 · Growth Measurement and Monitoring Days 0, 7, 14, 21 post-infection
  1. a Organoid imaging
  2. b Diameter measurement
  3. c Statistical analysis
Phase 4 · Organoid Harvest, Fixation, and Sectioning Days 3, 7, 14, 21 post-infection
  1. a Organoid isolation
  2. b PFA fixation
  3. c PBS washes
  4. d 15% sucrose equilibration
  5. e 30% sucrose equilibration
  6. f OCT embedding and freezing
  7. g Cryosectioning
  8. h Slide storage
Phase 5 · Immunofluorescence Staining for Cellular Architecture On day of staining
  1. a Slide preparation
  2. b Permeabilization
  3. c Blocking
  4. d Primary antibody incubation
  5. e PBST washes
  6. f Secondary antibody and nuclear staining
  7. g Final PBST washes
  8. h Mounting
  9. i Confocal microscopy imaging
  10. j Image processing
Phase 6 · Periodic Acid-Schiff (PAS) Staining for Fungal Detection On day of staining
  1. a Slide preparation
  2. b Periodic acid treatment
  3. c Distilled water washes
  4. d Schiff reagent treatment
  5. e Hot water rinse
  6. f Distilled water rinse
  7. g Light green counterstaining
  8. h Absolute alcohol rinses
  9. i Mounting
  10. j Microscopy imaging
  11. k Image processing
Phase 7 · Immunoblot Analysis for Cytokine Detection Days 3, 7 post-infection
  1. a Organoid collection
  2. b Accutase dissociation
  3. c Single cell suspension
  4. d Protein extraction
  5. e Lysate clarification
  6. f Protein quantification
  7. g Sample preparation for gel loading
  8. h SDS-PAGE gel electrophoresis
  9. i Protein transfer to membrane
  10. j Membrane blocking
  11. k Primary antibody incubation
  12. l Primary antibody washes
  13. m Secondary antibody incubation
  14. n Secondary antibody washes
  15. o Chemiluminescent detection
  16. p Film scanning and image processing
Phase 8 · RNA Isolation and Bulk RNA Sequencing Days 3, 7 post-infection
  1. a Organoid collection for RNA extraction
  2. b Organoid dissociation
  3. c Single cell suspension preparation
  4. d Lysis in RLT buffer
  5. e RNA extraction using Qiagen RNeasy minikit
  6. f RNA elution and quality assessment
  7. g Sample submission to sequencing facility
  8. h Sequencing and read generation
  9. i Raw read processing via RNA-seq Nextflow pipeline
  10. j Differential expression analysis (DESeq2)
  11. k Gene set enrichment analysis (GSEA)

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Attribution

This SOP was written by Organthis based on the published method in Harding et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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