Cryopreserved primary human hepatocytes

Pb and Pf infection of human PH was performed on either micropatterned co-culture (MPCC) preparations as previously described^{65 (link)} or on cultures of fresh primary human hepatocytes isolated from patients undergoing partial hepatectomy. Briefly, for micropatterned co-cultures, 1 × 10⁴ cryopreserved primary human hepatocytes (Life Technologies) were seeded on collagen-micropatterned plates softlithographically patterned with 500 µm islands together with 7 × 10³ 3T3-J2 murine embryonic fibroblasts. One day after seeding, 7 × 10⁴ freshly dissected Pb sporozoites were added to each well and subsequently, cells were fixed at various time points post infection for immunofluorescence microscopy analysis. For fresh primary human hepatocyte culture, viable hepatocytes were seeded into collagen-coated 96-well flat-bottom plates (5 × 10⁴ hepatocytes/well) in complete William’s B medium and cultured at 37 °C in an atmosphere of 5% CO₂. Two days after seeding, a batch of 5 × 10⁴ freshly dissected Pf or Pb sporozoites were added to each well. Cells were fixed 2 and 5 days after infection for Pb and for Pf, respectively. The number of infected hepatocytes was assessed by staining for Plasmodium Hsp-70 (mAb 2E6) and indirect immunofluorescence analysis.