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BREAST Publication-derived

Generation and Genetic Engineering of Human Breast Organoids from Donor-Derived Epithelial Cells

Source Walter and Eliza Hall Institute of Medical Research

👤 <UNKNOWN> ⏱ 35 days 📋 10 phases 🧫 Normal human breast epithelial cells from reduction mammoplasties

Abstract

This protocol describes the isolation, culture, and genetic engineering of three-dimensional breast organoids derived from normal human breast tissue. Normal mammary epithelial cells are sorted by flow cytometry and cultured in basement membrane extract to generate organoid structures. The organoids are subsequently transduced with lentiviral vectors encoding Cas9 and guide RNAs targeting tumor suppressor genes (P53, PTEN, RB1, NF1) to model breast cancer development.

Cell source
Normal human breast epithelial cells from reduction mammoplasties
Application
Disease modeling and tumor suppressor gene functional analysis

Protocol overview

56 steps across 10 phases

Human Breast Tissue Collection and Preparation Day 0-1
  1. 1 Obtain fresh human breast tissue
  2. 2 Chop breast tissue prior to enzymatic digestion
  3. 3 Enzymatic digestion of breast tissue
  4. 4 Wash and centrifuge digested cells
  5. 5 Trypsin treatment to obtain single cell suspension
  6. 6 Filter single cell suspension
Flow Cytometry Sorting of Epithelial Cells Day 1
  1. 1 Block and incubate with primary antibodies
  2. 2 Wash cells and incubate with streptavidin
  3. 3 Prepare for flow cytometry
  4. 4 Sort cells on FACSAria flow cytometer
Organoid Culture Initiation Day 2–14
  1. 1 Seed sorted cells in basement membrane extract
  2. 2 Prepare organoid culture medium
  3. 3 Culture organoids
  4. 4 Refresh culture medium
  5. 5 Passage organoids
Lentiviral Vector Production Day X (prior to organoid transduction)
  1. 1 Transfect HEK293T cells with lentiviral plasmids
  2. 2 Refresh media post-transfection
  3. 3 Collect viral supernatant
  4. 4 Concentrate viral supernatant
  5. 5 Determine virus titers
Organoid Transduction and Genetic Editing Day 7–35 (corresponding to organoid passages 1–5)
  1. 1 Dissociate organoids prior to first transduction
  2. 2 Prepare cells for transduction
  3. 3 Incubate cells with virus
  4. 4 Wash transduced cells
  5. 5 Reseed transduced cells in BME
  6. 6 Select for P53-edited cells (first round)
  7. 7 Perform second transduction with RB1 sgRNA
  8. 8 Perform third transduction with NF1 sgRNA
  9. 9 Select for NF1-edited cells
Organoid Characterization and Quality Control Throughout culture (Days 2–35+)
  1. 1 Perform immunofluorescence staining of organoids
  2. 2 Incubate with secondary antibodies and stains
  3. 3 Clear organoids prior to imaging
  4. 4 Perform 3D confocal imaging
  5. 5 Perform 3D image reconstruction
Organoid Transplantation into Mice After genetic editing is complete (passages 3–5+)
  1. 1 Prepare organoids for transplantation
  2. 2 Prepare transplantation mixture
  3. 3 Transplant organoids into mice
  4. 4 Monitor transplanted mice
  5. 5 Harvest tumor tissue
Tumor Tissue Analysis At endpoint post-transplantation
  1. 1 Fix tumor tissue
  2. 2 Embed tumor tissue in paraffin
  3. 3 Perform antigen retrieval
  4. 4 Immunohistochemistry staining
  5. 5 Secondary antibody incubation
  6. 6 Signal detection
  7. 7 Counterstaining and mounting
Validation in MCF10A Cells (Optional Quality Control) Prior to or in parallel with organoid editing
  1. 1 Culture MCF10A cells
  2. 2 Transduce MCF10A cells with viral vectors
  3. 3 Sort fluorescent cells
  4. 4 Culture sorted cells
  5. 5 Western blot analysis
  6. 6 Antibody probing
Next-Generation Sequencing Validation (Optional) At endpoint or after genetic editing
  1. 1 Design and prepare PCR primers
  2. 2 Perform PCR amplification
  3. 3 Prepare PCR products for sequencing
  4. 4 Sequence and analyze results

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in the published method. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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