Source: Finney, C. et al., An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection. J. Vis. Exp. (2015)
This video demonstrates the generation of an in vitro malaria-HIV co-infection cell culture model. HIV-infected peripheral blood mononuclear cells co-cultured with Plasmodium falciparum parasitized erythrocytes potentially support each other's growth and provide insights into the immune responses and disease progression in HIV and malaria co-infections.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
CAUTION: Working with human blood samples and human malaria parasites requires precautionary measures. Always wear a lab coat, and gloves, and work in a level 2 Biosafety cabinet. In the event of accidental percutaneous exposure to human malaria, report to Health and Safety for prophylactic treatment. Additional safety considerations should also be put in place for working with human immunodeficiency virus HIV(+) blood. Wear a back-closing lab coat. Double glove (top glove should be latex). Perform all handling in a level 2 Biosafety cabinet. Do not use any glass or sharp objects. Do not use an aspirator. Place all contaminated items in virox solution or bleach for a minimum of 1 hr prior to discarding. Wash all surfaces with virox and ultraviolet (UV) for 1 hr following use. Note that each institution will have its own specific biosafety regulations that need to be followed. Report all accidental exposures to HIV-infected blood to Health and Safety for evaluation and consideration of possible post-exposure prophylaxis.
Note: Different strains of P. falciparum malaria parasites exist. ITG was used for these experiments, but different strains can be used. Excellent instructions on freezing and thawing Plasmodium falciparum parasites are available at the MR4 website.
1. Making RPMI-A for Malaria Parasite Culture
2. Preparing Human Red Blood Cells for Parasite Culture
Note: Blood donors should be type O.
3. Maintaining Parasite Cultures
4. Parasite Synchronization
Note: The day before the experiment, synchronize the parasite culture by treating it with alanine. Only ring-stage parasites and uninfected RBCs will survive this treatment. Alanine synchronization will give you a pure trophozoite culture the next day that can be used in the co-culture experiments. Make sure to start with a parasite culture that contains a majority of ring-stage parasites.
5. Parasite and RBC Preparation for Co-culture Experiments
6. Isolation of Human Peripheral Blood Mononuclear Cells (PBMC)
7. Malaria/HIV Co-infection Culture
8. Detection of Malaria Immune Responses
Note: Perform co-culture experiments for as long as 4 days. No medium change is required during this time. The optimal time point will depend on the cell type of interest and the question asked. An incubation of 12-48 hr is optimal for monocytic responses to PfRBCs, while lymphocyte responses were best observed at 72-96 hr. The time points will need to be optimized based on the experimental question. Shorter periods (2-4 hr) may be used if the interaction between intact PfRBC and PBMCs is of interest.
Figure 1. Depiction of Ficoll gradient post spin demonstrating the position of the PBMCs.
The authors have nothing to disclose.
Alanine | Sigma | A7377 | |
BD Vacutainer ACD Solution A | BD | 364506 | |
BD Vacutainer Sodium Heparin | BD | 17-1440-02 | |
DPBS (no calcium, no magnesium) | Corning | 21-031-CV | |
Fetal Bovine Serum | Sigma | F1051 | Heat inactivate before use |
Ficoll-Paque PLUS | GE Healthcare | 17-1440-02 | |
Gentamycin (10mg/ml) | Gibco | 15710-064 | |
Hema3 Staining Set | Fisher | 122-911 | |
HEPES | Fisher | BP310-500 | |
Hypoxanthine | Sigma | H9636 | |
Ionomycin | Sigma | I3909 | |
MEM non-essential amino acids (10mM) | Gibco | 11140 | |
PMA | Sigma | P8139 | |
RPMI-1640 powder | Life-Technologies | 31800-022 | |
RPMI-1640 with L-glutamine and HEPES | Thermo Scientific | SH30255.01 | |
Sodium bicarbonate (powder, cell culture) | Sigma | S5761 | |
Sodium Pyruvate (100mM) | Gibco | 11360 | |
Tris (Trizma base) | Sigma | T6066 | |
Trizol | Ambion | 15596018 | |
Trypan Blue (0.4%) | Gibco | 15250-061 | |
Parasite Gas Mixture | By special order | 3% CO2, 1% O2, balance N2 |