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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

From: From marginal to essential: the golden thread between nutrient sensing, medium composition and Plasmodium vivax maturation in in vitro culture

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, 115,116,117,118,119,120,121,122,123]

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

[179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194]

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

[167,168,169,170,171,172]

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]

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