Biopreservation of RBCs for in vitro Plasmodium falciparum culture

Falciparum malaria remains a devastating infectious disease, killing nearly 700,000 people annually. Further understanding of malaria pathogenesis will help identify molecular and cellular targets of next-generation therapeutics. The morbidity and mortality of malaria infection occur during the erythrocytic stage. Study of erythrocytic stage malaria is critical not only for new anti-malarial development, but also for increasing our understanding of the host pathogen relationship. For example, many host genetic polymorphisms known to impact red blood cell (RBC) physiology also alter malaria susceptibility.

Methods for culturing erythrocytic stage P. falciparum were developed more than 30 years ago (Jensen & Trager, 1977). However, optimization of in vitro P. falciparum culture is still under investigation. The aspects of intraerythrocytic development that are impacted by RBC storage, as well as the effects of RBC biopreservation on the intraerythrocytic life cycle, remain unknown.

Studies on RBC storage for human clinical use reveal a relationship between RBC storage and transfusion complications (Aubron et al, 2013). Current blood banking standards involve RBC storage in SAGM (a solution containing saline, adenine, mannitol and glucose) or closely related solutions for up to 42 days at 4°C (Sparrow, 2012). Many RBC storage lesions have been documented in these acidic medias, including decreased intracellular ATP, 2,3-diphosphoglycerate (2,3-DPG) and potassium; increased intracellular NaCl; oxidative damage; lipid peroxidation; membrane phospholipid changes and vesiculation; decreased deformability; reduced glycolytic capacity; and decreased vasodilatory capacity and increased cytoadhesion (Aubron et al, 2013; Bennett-Guerrero et al, 2007). Overall, these storage lesions are similar to physiological changes occurring with normal RBC ageing in the bloodstream (Franco et al, 2013) and could also impact parasite growth, as P. falciparum preferentially infects younger RBCs in circulation (Lim et al, 2013; Clark et al, 2014a).

The use of biopreserved RBCs for human transfusion has been validated (Fabricant et al, 2013) and cryopreserved umbilical cord blood cells can propagate P. vivax (Borlon et al, 2012) Here we examine the impact of RBC storage and biopreservation on P. falciparum growth and development in vitro. Prolonged RBC shelf-life and biopreservation could enhance malaria research by: (1) enabling standardization of the RBC source for multiple experiments, and (2) increasing access to RBCs from individuals with unusual blood types, nutritional deficiencies, or from remote locations.

Given lingering discrepancies in standard P. falciparum culture protocols, we sought to definitively assess RBC shelf life. To begin, fresh RBCs were collected into acid citrate dextrose (ACD) and stored in aliquots as packed RBCs at 4°C for up to 6 weeks. At 2-week intervals, fresh RBCs were obtained and identically stored to allow for simultaneous comparisons of parasite growth in vitro using blood stored for 0, 2, 4 and 6 weeks. Growth in RBCs stored for 2 weeks showed no decrease in standard 96-h growth assays (Clark et al, 2014a). After 4 weeks of storage, growth rates diminished significantly (42% decline for Dd2, 65% for FCR3). In RBCs stored for 6 weeks, there was very little growth (over 90% decline for both strains Dd2 and FCR3) (Figure 1A).

Figure 1. Effects of RBC storage conditions on P. falciparum growth in vitro.

A. Growth assays in RBCs stored 0, 2, 4, or 6 weeks in ACD at 4°C. Growth rates in stored RBCs are normalized to fresh RBCs. Error bars represent the standard deviation (SD). *p<0.01, compared to growth rates in fresh RBCs. B. Growth rates in RBCs drawn into ACD and packed were either stored as is, or in alternative storage media (Alsever’s, ACD, CPDA, RBC Buffer). *p<0.01, compared to growth rates in fresh RBCs. (RBC Buffer = 10 mM HEPES, 12 mM NaCl, 115 mM KCl, 5% BSA). C. PEMR in RBCs stored 0, 2, and 4 weeks. PEMR is normalized to that in fresh RBCs. Error bars represent the SD. D. Susceptibility Index (SI) for fresh RBCs compared to RBCs stored for 0, 2, 4 and 6 weeks. An SI of 1.0 indicates no invasion difference between two RBC populations. The marker represents the SI point estimate and the bars represent the 95% confidence intervals.

RBCs, red blood cells; ACD, acid citrate dextrose; CPDA, citrate-phosphate-dextrose-adenine; PEMR, parasite erythrocyte multiplication rate; Dd2, P. falciparum Dd2 strain; FCR3-FMG, P. falciparum FCR3-FMG strain

We next compared parasite growth in RBCs stored for 0, 2 and 4 weeks at 4°C in four different storage buffers. Buffers tested were: (1) ACD; (2) citrate-phosphate-dextrose-adenine (CPDA), commonly used for malaria culture; (3) Alsever’s Solution, a balanced salt solution routinely used for RBC washing prior to parasite culture; (4) “RBC buffer” an alternative balanced salt solution. Growth rates decreased proportionally to storage length in each of the buffers, with a significant decrease after 4 weeks of storage (62% for ACD, 56% for CPDA, 54% for Alsever’s and 51% for “RBC buffer”) (Figure 1B). This confirms RBCs destined for parasite culture must be used within 2 weeks of collection and that differential storage media does not prolong their shelf life for P. falciparum culture.

We next sought to determine whether parasite replication and/or invasion were decreased in stored RBCs. To assess replication, we measured the parasite erythrocyte multiplication rate (PEMR), which reflects the number of infectious merozoites produced per schizont (Clark et al, 2014a). We found no statistically significant differences in replication between RBCs stored for 0–4 weeks (Figure 1C). Invasion rates were assayed using a RBC barcoding assay (Clark et al, 2014b) in which differentially labelled RBCs (with CellTrace membrane dyes; Life Technologies Corp., Grand Island, NY, USA) were combined in the same wells and seeded with unlabelled trophozoite stage parasitized RBCs. This assay allows direct comparison of parasite invasion into two different RBC populations. Invasion rates decreased as RBC storage time increased (Figure 1D). We hypothesize that this reduction in invasion is due to decreases in intracellular ATP content and RBC deformability, which are associated with RBC storage. The use of extensively stored RBCs could artificially indicate growth differences between donors with divergent RBC phenotypes, when, in reality, differential RBC storage length might primarily contribute to any observed difference. Routine use of out-dated RBCs could also result in the artificial selection of strains that preferentially invade atypical RBCs.

In an effort to increase the shelf life of RBCs for malaria culture, we turned to biopreservation. RBC freezing/thawing resulted in minimal RBC lysis (Figure 2A). Biopreserved RBCs were able to support equally high rates of P. falciparum growth as fresh RBCs (Figure 2B). No variation of washing before or after freezing/thawing consistently increased biopreserved RBC shelf life. Successful merozoite invasion of biopreserved RBCs was confirmed using the RBC barcoding assay. No differences were observed between invasion of fresh versus biopreserved RBCs (Figure 2C).

Figure 2. Biopreserved RBCs are suitable for P. falciparum growth.

(A) Biopreserved RBCs show minimal lysis after deglycerolization and centrifugation. (B) Comparison of growth rates between fresh RBCs and biopreserved RBCs differentially washed. To freeze RBCs, 200µl of fresh RBCs were mixed with 300µl human plasma, then 500µl freezing media (28% glycerol, 3% sorbitol, 0.65% NaCl) was added and cells were frozen in liquid nitrogen. To thaw, 200µl of 12% NaCl was added to the frozen RBCs, incubated for 5 minutes and pelleted by centrifugation. Subsequently, RBCs were washed with 1.6% NaCl, followed by 0.9% NaCl and used immediately for culture. Variations tested included: washing with Alsever’s Solution (BioP-1) or parasite culture media (BioP-2) three times prior to freezing. Cells pre-washed in Alsever’s solution were either used directly (BioP-1) or rewashed three times after thawing in Alsever’s solution (BioP-3). Error bars represent the standard deviation. (C) Susceptibility Index (SI) for fresh versus biopreserved RBCs. The marker represents the SI point estimate and the bars represent the 95% confidence interval.

Our finding that biopreserved RBCs are suitable for P. falciparum assays has vast practical applications. Using biopreserved RBCs minimizes the need to obtain fresh blood, as fresh RBCs could be frozen in aliquots and individually thawed when needed. Our biopreservation method could also facilitate field studies in areas where immediate parasite growth is difficult. Additional studies are needed to determine if this technique can be applied to ‘variant’ RBCs, such as RBCs from individuals with sickle cell trait or glucose-6-phosphate dehydrogenase deficiency. Freezing RBCs also allows for extensive evaluation of parasite growth in unique blood types and standardization in gene expression assays.