Table 2.

Proposed systematic comparisons needed to unravel key avenues for the development of a continuous in vitro culture system for P. vivax blood stages

Media component	Proposed systematic comparisons with current practice	Expected effect on the parasite	Expected effect on the reticulocyte	Media formulation to accomplish proposed action	References
	Earle’s salts + reduce sodium bicarbonate to 0.85 g/L	Optimal buffering capacity: parasite healthy maturation	Reticulocyte healthy maturation	RPMI Dutch’s modification	Never tested
	amino acids + mixture of sugars; no sodium bicarbonate	Optimal buffering capacity: parasite healthy maturation	Reticulocyte healthy maturation	L15 and SR1-8	[106]
	HEPES 20 mM	Optimal buffering capacity: parasite healthy maturation	Reticulocyte healthy maturation		[108]
Inorganic salts	Ionic strength of 12.6–12.80 mS/cm	Better segmentation	Reticulocyte healthy maturation	Waymouth	[86]
	Increase Magnesium chloride 1.8 mM	Increased parasite viability	Appropriate conformation of surface proteins	Waymouth/L15	[86]
	Addition of calcium pantothenate	Enhanced parasite survival and multiplication	If any, unknown	SCMI 612	[40]
	Addition of manganese	Enhanced parasite invasion	Appropriate conformation of surface proteins		[111, 112]
	GlutaMAX	Does not break down to form toxic ammonia: better progression to late stages	Reticulocyte healthy maturation	Commercial McCoy5A/RPMI commercial + glutamax	[35]
	Increase leucine, phenylalanine, valine, isoleucine and methionine	Parasite growth and detoxification of the toxic free heme	Healthy reticulocytes	In most media formulations; concentration may vary	[12, 103, 106, 115–123]
Amino acids	Ascorbic acid	Enhanced parasite survival and multiplication	Healthy reticulocytes: helps to keep proper redox equilibrium	In most media formulations; concentration may vary	[86]
	Orotic acid	Enhances parasite redox stability	Healthy reticulocytes: helps to keep proper redox equilibrium	Needs to be added, concentration to be determined	[59]
Vitamins	pABA concentrations	Enhanced intracellular development and maturation	Healthy reticulocytes	Most appropriate concentration to be determined	[31, 128.130]
	Piridoxyne/pyridoxal hydrocholoride	Enhanced intracellular development and maturation	Healthy reticulocytes	IMDM has highest concentration at 4 g/L	[127, 142, 144]
Serum	Use serum (ideally commercial); serum is likely better than Albumax	Supporting parasite viability	Membrane integrity and maturation	Commercial serum is desirable for inter laboratory standardization	[179–194]
Antibiotics	Gentamicin/other antibiotics	Possible toxicity on specific parasite species and strains	If any, unknown		[64]
Hypoxanthine	Necessary	Parasite maturation	If any, unknown	Waymouth, DMEM/F12	[36]
Lipid	Addition of fatty acids, choline, myo-inositol, ethanolamine, lyso-PC	Parasite survival, maturation and multiplication	Membrane integrity and maturation	Needs to be added, concentration to be determined	[167–172]
Cholesterol	Use lipoproteins containing cholesterol instead of free cholesterol	Parasite maturation and eggress	Membrane integrity and maturation	Needs to be added, concentration to be determined	[175]

Expected effects of proposed approaches on both the parasite and the reticulocyte