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LIVER Publication-derived

Liver 2D Cell Cultures for Drug Screening

Full SOP with Academic or a plan

Source Ng et al., 2015 · Massachusetts Institute of Technology · 10.1016/j.stemcr.2015.01.002

Authors: Shengyong Ng, Robert E. Schwartz, Sandra March, Ani Galstian, Nil Gural, Jing Shan, Mythili Prabhu, Maria M. Mota, Sangeeta N. Bhatia Duration: 28 days 7 phases Cell source: Human iPSC

Abstract

This protocol describes the differentiation of human iPSCs into hepatocyte-like cells (iHLCs) and their use as an in vitro model for Plasmodium liver-stage infection. iHLCs support infection by multiple Plasmodium species (P. berghei, P. yoelii, P. falciparum, P. vivax) and can be chemically matured to improve drug metabolism capabilities for antimalarial drug testing.

Cell source
Human iPSC
Application
Disease modeling; Antimalarial drug screening

Protocol overview

34 steps across 7 phases

Phase 1 · iPSC Culture and Hepatocyte-like Cell Differentiation 0-20 days
  1. a Culture iPSCs on Matrigel
  2. b Initiate directed differentiation with Activin A (Day 0-5)
  3. c Apply bone morphogenetic protein 4 and basic FGF (Day 5-10)
  4. d Treat with HGF to generate hepatoblasts (Day 10-15)
  5. e Complete maturation with oncostatin M (Day 15-20)
Phase 2 · Preparation of Plasmodium Sporozoites Prior to infection
  1. a Obtain infected mosquitoes
  2. b Dissect salivary glands
  3. c Prepare sporozoite suspension
Phase 3 · Infection of iHLCs with Plasmodium Sporozoites Day 20+ post-differentiation
  1. a Prepare iHLC cultures
  2. b Centrifuge sporozoites onto iHLCs
  3. c Incubate for infection
  4. d Wash and replace medium
  5. e Daily medium replacement
  6. f Fix infected cultures at appropriate timepoints
Phase 4 · Immunofluorescence Assay for Parasite Detection and Quantification Following fixation
  1. a Wash fixed cultures with PBS
  2. b Block with BSA
  3. c Incubate with primary antibodies
  4. d Wash with PBS
  5. e Incubate with secondary antibodies
  6. f Counterstain nuclei
  7. g Final wash and mount
  8. h Image and analyze
Phase 5 · Antimalarial Drug Testing During infection (Day 20+ post-differentiation)
  1. a Add antimalarial drugs at infection
  2. b Maintain drug treatment
  3. c Fix and analyze drug-treated cultures
Phase 6 · Small Molecule-Mediated Maturation of iHLCs (Optional) Days 21-28 post-differentiation
  1. a Treat iHLCs with FPH1 (maturation phase)
  2. b Washout period
  3. c Infect matured iHLCs
  4. d Assess primaquine sensitivity in matured iHLCs
Phase 7 · Characterization of iHLC Hepatic Markers Days 0-20 post-differentiation (in parallel with Phase 1)
  1. a Collect cell culture supernatants
  2. b Measure hepatic protein secretion by ELISA
  3. c Assess CD81 and SRB1 gene expression
  4. d Assess CD81 and SRB1 protein expression
  5. e Assess CD81 and SRB1 cell surface localization

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Attribution

This SOP was written by Organthis based on the published method in Ng et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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