Step-by-Step Guide: Culturing Plasmodium falciparum Strains in the Lab

 

Culturing Plasmodium falciparum Strains in the Lab 

Culturing Plasmodium falciparum in the lab is a critical process for malaria research. This article provides the instructions for culturing laboratory strains of Plasmodium falciparum, the deadliest species of malaria parasite.
The cells are typically cultured in vitro using human erythrocytes (red blood cells, RBCs). The culture system requires precise control of environmental conditions, including temperature, gas composition, and nutrient availability.
For a detailed walkthrough of this protocol, check out my YouTube video embedded below!  

Click to watch my video on culturing P. falciparum strains!

Disclaimer 

Culturing Plasmodium falciparum is a highly specialized technique that requires extensive training, biosafety level 2 or 3 containment, and access to specialised equipment.
Culturing Plasmodium parasites, including Plasmodium falciparum, requires strict adherence to safety precautions due to the risks associated with handling pathogens and human blood products. Key safety measures include:

1. Biosafety Level 2 (BSL-2) Facilities: Labs must operate under BSL-2 conditions when working with Plasmodium cultures. This includes the use of biological safety cabinets for procedures that generate aerosols, proper ventilation, and restricted access to the laboratory to limit exposure.
2. Personal Protective Equipment (PPE):  Laboratory personnel should wear gloves, lab coats and eye protection. additional protective measures, such as face shield, may be necessary when handling cryopreserved samples or during procedures involving liquid nitrogen.
3. Handling of Human Blood: Since human red blood cells are used in the culture medium, there is a risk of transmitting bloodborne pathogens. All blood samples should be treated as potentially infectious, sourced from accredited suppliers, and screened for pathogens like HIV and hepatitis.
4. Decontamination Procedures: Spills, waste, and contaminated materials should be decontaminated using appropriate disinfectants, such as 10% bleach or autoclaving. Waste disposal must follow regulatory guidelines for biohazardous materials.
5. Cryopreservation Risks: When thawing cryopreserved Plasmodium samples, handle under sterile conditions to prevent cross-contamination.
6. Aerosol Generation Control: Manipulations that could generate aerosols should be minimised, as they pose a risk of infection. Procedures such as centrifugation must use sealed rotors or safety cups to contain aerosols.
7. Training and Documentation: Personnel must be trained in handling infectious agents and in emergency protocols. Laboratory procedures should be well-documented, and safety data sheets for all chemicals used must be readily accessible.

Preparing the Culture Medium  

1. Preparation of InComplete RPMI Culture Media From Powder Stock

For the Plasmodium falciparum cell line, the media that is used for growth is known as Complete RPMI Culture Media. 500mL of the InComplete RPMI culture media can be prepared as follows
  - 7.94g of powdered RPMI-1640
  - 424mg of Sodium Bicarbonate (NaHCO3)
  - 50mg of Hypoxanthine in 1mL of 1M NaOH
  - 500ul of Gentamicin
  
Make up to 500mL with distilled water.
Filter via a 0.22um filter to ensure sterility before use.

You may also purchase pre-made liquid RPMI-1640 from the vendors below:

• Sigma Aldrich: Website: Sigma Aldrich.
• Lonza. Website: Lonza
• VWR International: Website: VWR International
• Corning (via VWR or Direct Supply): Website: Corning

2. Preparation of Complete RPMI Culture Media 

Prepare Albumax by adding 5g to 100mL of distilled water. Autoclave before addition to media.  
Make Complete media by adding the following:
  - 45mL of RPMI
  - 5ml of autoclaved Albumax (replaces blood serum or plasma) 
  - 1.3mL of NaHCO3

Tightly cap the RPMI-1640 primary container and gently invert 5 times to mix.
Store at 4oC when not in use.
Label with "Complete media," Expiry (1 month from preparation) and your initials.

3. Erythrocyte (RBCs) Preparation

For the parasite cell line, the cell culture media is Complete media supplemented with 5% RBCs.
Prepare erythrocytes to a final haematocrit of 50% as follows:  

1.  Collect human blood from a compatible donor.
2.  Separate RBCs from plasma and buffy coat by centrifugation.
3. Wash RBCs x3 times with sterile Phosphate Buffered Saline (PBS) to remove plasma proteins. 
4. Resuspend RBCs in culture medium to a working stock of 50%

Resuspend RBCs in parasite culture to a final haematocrit of 5%.

4. 10% Giemsa Preparation

Perform a 1:10 dilution of the stock 100% Giemsa by adding 1mL of Giemsa reagent to 9mL Phosphate Buffered Saline (PBS).
Filter the 10% solution using a 0.22um filter.

5. Culture Initiation 

1.   Thaw frozen parasite stock (See Appendix 1 below, for how to thaw Plasmodium parasite stocks).  
2.   Add thawed synchronised parasites to the prepared RBC culture.
3.   Gas the culture for 20-30 seconds using a mixture of 5% carbon dioxide (CO2), 5% Oxygen (O2) and 90% Nitrogen (N2). Cap the culture vessel tightly and incubator at 37oC.

Culture Maintenance

1. Monitor parasite growth daily using microscopy.
2. Replace the culture medium with fresh Complete media
3. Maintain hematocrit by adding fresh RBCs every 2-3 days to maintain parasite growth

6. Parasite Quantification

Thin smear of parasite culture. Image credit: Yakubu Osmanu (Noguchi Memorial Institute of Medical Research)

Determine parasite density and parasitaemia using microscopy:

• To do this, count a minimum of 500 and a maximum of 1000 RBCs (obtain these minimum and maximum counts by dividing your field of view into 4 quadrants. Count the number of RBCs in one quadrant and multiply it by x4).

Fig. 1a : An example of a typical microscopic view of a thin blood smear from the culture. Ring Stage parasites appear with a dark purple dot (the nucleus), with a lighter purple cytoplasm surrounding it. Trophozoites appear much larger with concentrated purple staining. In later stages, you will see multiple nuclei, representing the multiple parasites of the Schizont stage.

1. Count the number of parasites at ring, trophozoite and gametocyte stages of development, inside the counted RBCs.
2. Determine parasitaemia by taking the sum of all parasites counted (across the various developmental stages) and divide the number by the total number of RBCs.
3. Multiply (x) the results by 100 to obtain the percent parasitaemia.

Subculture the parasites when you obtain parasitaemias above 4%. Do not exceed 5% parasitaemia.

NB: Sometimes, you may have purple indications inside RBCs which may not be parasites. See Appendix 2 for pseudo staining that you should not count as parasites.

Appendix 1 - Thawing Plasmodium Parasite Stocks From -80oC Freezer or Liquid Nitrogen Storage.

Preparation of 12% (Solution A), 1.6% (Solution B) and 0.9% (Solution C) Sodium Chloride (NaCl) Solutions

Prepare 10ml of each of the required NaCl solutions by adding 1.2g (Solution A), 0.16g (Solution B) and 0.09g (Solution C) NaCl to distilled water. Filter with a 0.22um filter to ensure sterility. 

Thaw the frozen vial of plasmodium cells as follows:

1. Remove a vial from liquid nitrogen or -80oC freezer and thaw at 37oC immediately.
2. Transfer the thawed parasites into a 10ml sterile tube to determine the final volume of the parasites. Note down the volume.
3. Using a syringe, aspirate 0.2X volume of solution A in reference to the thawed parasite volume. For example, if the volume of the thawed parasites is 0.5ml, take 0.1ml of solution A and add this drop-wise to the thawed parasites. Use a pasteur pipette to gently mix solution A and the parasites.
4. Allow the solution A/parasite mix to stand at room temperature for 2 minutes to draw out the freezing mixture.
5. Next, measure 10X the volume of solution B in a 5ml syringe. For example, if the volume of the parasite/solution A is 0.5ml, measure 5ml of solution B. Slowly add this to the parasite mixture and mix gently using a pasteur pipette.
6. Centrifuge the solution for 5 minutes at 1500 x g.
7. Remove the supernatant without disturbing the red cell layer.
8. Measure 10X the volume of solution C in a 5ml syringe and add thes slowly to the pelleted parasites. Mix gently using a pasteur pipette.
9. Centrifuge again for 5 minutes at 1500 x g.
10. Remove the supernatant carefully, making sure that the red blood cells remain in the tube ready for culture initiation.

Appendix 2 - Pseudo purple stains that may be confused for parasites inside RBCs. Notice the lack of distinct staining of the nucleus.

Bibliography

1. Trager, W. & Jensen, J.B., 2005. Human malaria parasites in continuous culture. The Journal of Parasitology, 91(3), pp.484-486. DOI: 10.1645/0022-3395(2005)091[0484:HMPICC]2.0.CO;2.
2. Doolan, D.L. ed., 2008. Malaria methods and protocols (Vol. 72). Springer Science & Business Media.
3. Radfar, A., Méndez, D., Moneriz, C., Linares, M., Marín-García, P., Puyet, A., Díez, A. & Bautista, J.M. (2009) 'Synchronous culture of Plasmodium falciparum at high parasitemia levels', Nature Protocols, 4(11). Available at: https://doi.org/10.1038/nprot.2009.198.

December 03, 2024 Tags : Continuous parasite culture , Culturing malaria parasites , Erythrocyte cultures for malaria , Malaria research techniques , Plasmodium falciparum culturing

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