Skip to content
Back to browse
LIVER Publication-derived

Malaria Liver Spheroids

Full SOP with Academic or a plan

Source Chua et al., 2019 · Novartis Institute for Tropical Diseases, Singapore · 10.1016/j.biomaterials.2019.05.032

Authors: Adeline C.Y. Chua, Abhishek Ananthanarayanan, Jessica Jie Ying Ong, Jen Yi Wong, Andy Yip, Nisha Hari Singh, Yinghua Qu, Laurent Dembele, Michael McMillian, Ratawan Ubalee, Silas Davidson, Anchalee Tungtaeng, Rawiwan Imerbsin, Kapish Gupta, Chiara Andolina, Fan Lee, Kevin S-W Tan, François Nosten, Bruce Russell, Amber Lange, Thierry T. Diagana, Laurent Rénia, Bryan K.S. Yeung, Hanry Yu, Pablo Bifani 6 phases Cell source: Human primary hepatocytes; Cynomolgus monkey primary hepatocytes

Abstract

This protocol describes the development and use of 3D spheroid-cultured primary hepatocytes in a Cellusponge scaffold to model the complete malaria liver stage cycle of P. vivax and P. cynomolgi, including hypnozoite reactivation and relapse. The system supports infection with sporozoites, parasite maturation, and merozoite invasion of erythrocytes, enabling predictive screening of antimalarial compounds for causal prophylactic versus hypnozoitocidal activity.

Cell source
Human primary hepatocytes; Cynomolgus monkey primary hepatocytes
Application
Disease modeling; Drug screening for malaria radical cure compounds

Protocol overview

30 steps across 6 phases

Phase 1 · Hepatocyte Culture and Spheroid Formation Day 0-4
  1. a Prepare hepatocyte culture medium
  2. b Thaw cryopreserved hepatocytes
  3. c Seed hepatocytes onto 3D Cellusponge
  4. d Add supplemental medium
  5. e Monitor spheroid formation
Phase 2 · Parasite Infection (Pre-formed Spheroid Method) Day 4-6
  1. a Prepare sporozoites
  2. b Infect pre-formed spheroids
  3. c Wash infected spheroids
  4. d Maintain infected spheroids
Phase 3 · Parasite Infection (Suspension Method) Day 0-4
  1. a Prepare sporozoites
  2. b Co-incubate hepatocytes with sporozoites in suspension
  3. c Collect and wash infected hepatocytes
  4. d Seed infected hepatocytes onto 3D Cellusponge
  5. e Maintain infected spheroids
Phase 4 · Drug Treatment Models Day 0-21
  1. a Prepare drug solutions
  2. b Implement Causal Prophylaxis Model (Days 0-5)
  3. c Implement Delayed-Treatment Model (Days 4-10)
  4. d Implement Radical Cure Model (Days 4-16, then 5-day release)
  5. e Fix infected spheroids for analysis
Phase 5 · Immunostaining and Imaging Day 5, 10, or 21 (dependent on phase)
  1. a Wash fixed spheroids
  2. b Block non-specific binding
  3. c Incubate with primary antibodies
  4. d Wash and incubate with secondary antibodies
  5. e Apply DAPI nuclear stain and mount
  6. f Acquire confocal images
  7. g Analyze fluorescence intensity
Phase 6 · Blood Stage Invasion Assay (Optional) Day 7-21
  1. a Collect erythrocytes
  2. b Overlay erythrocytes on infected spheroids
  3. c Sample and stain blood
  4. d Record parasitemia and morphology

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Chua et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence