According to the experience of some colleagues of mine in this field, promastigotes from Leishmania spp growht well in RPMI supplemented with heat-inactivated FCS, glutamine and antibiotics. 

Axenic amastigotes are cultured using the Schneider’s medium at ph 5.0. Maybe the following work may help you:

http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0001612#pntd-0001612-g006

Leishmania promastigotes can grow easily in a variety of culture media (RPMI 1640, Schneider, Grace, M199, among others) with FCS + supplements at 26 ºC. in my experience there are differences among species, isolates and strains to get the maximum growth. I wopuld suggest to test, for a particular strain, some of them to select the optimum culture medium for your particular conditions.

According to the experience, for some Leishmania stocks or WHO reference strains, it is necessary to use USMARU médium with PBSS plus antibiotics to recover and develop the promastigotes culture, before to transfer to a liquid medium, such as RPMI or Schneider. You have a detailed protocol in Marco et al, Am J Trop Med Hyg. 2005 May;72(5):606-11.

Thanks for the paper. I found it with enough details

The attached papers might be useful for you.

Hope it helps.

Lemesre J.L., Kweider M., Darcy F. and Santoro F (1988). Requirement of defined cultivation conditions for standard growth of Leishmania  promastigote in vitro. Acta Tropica, 45: 99-108.

Merlen T, Sereno D, Brajon N. and Lemesre J.L. (1999). Leishmania Spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms. Am. J. Trop. Med. Hyg., 60 (1), 41-50.

All culture media mentioned above are suitable for the cultivation of Leishmania spp. In our experience we use Schneider's or RPMI supplemented with 5% or 10% heat inactivates FCS + 2% human urine and antibiotics and growth at 25-26o C.

The culturing of the Leishmania parasites is done in Corning 25cm2 tissue culture flasks (non-vented caps) containing 10 mL of the growth medium principally RPMI-1640 or Medium-199. This medium is complemented with 10% heat-inactivated Fetal Bovine Serum and 1% the of penicillin-streptomycin solution. The parasites are cultured in an incubator for a week, with regular observations for growth under an inverted microscope. The incubation is under anaerobic conditions and at 24-26oC. The culture is observed for parasitic density by using Neubauer Haemocytometer. Once a bulk of promastigote reach, the culture is sub-passaged (at late-log stage of the growth) into fresh growth medium to a dilution of 1×105/mL. Promastigotes density in the culture is verified by using Neubauer Haemocytometer, and in this way the parasites are harvested for the succeeding assay. The unused parasites (if more than 5×106/mL) are cryopreserved by using cryomedium (40% RPMI+7.5% DMSO or 10% Glycerol+50% FBS) first in (Freezing container, Nalgene®) Mr. Frosty at -20oC (requires 100% isopropyl alcohol and provide repeatable minus -1°C/minute cooling rate for successful cryopreservation) and then at -86oC in ultrafreezer or at -196oC in in liquid nitrogen for later used.

I used SDM 97 medium supplemented with 10% FCS and hemin

Culture conditions and Sub-passaging

The cutaneous Leishmaniasis exudates are cultured in M-199 growth medium (Table 1) supplemented with 5µg/ml of Hemin and 10% heat inactivated (HI-FBS) fetal bovine serum. An equal volume of RPMI 1640 (Sigma R5886) is then added. After bulking up Leishmania promastigote densities of 1x105/ml are cultured in the resulting medium and incubated in closed tissue-culture flasks (Sterilin) at a constant temperature of 24-26°C. Promastigotes are subpassaged when they reach the late-log stage of growth into fresh growth medium to a dilution of 1x105/mL.

Estimation of promastigote density

Cell density is verified daily by light microscopy and the use of an Improved Neubauer haemocytometer. These observations allow us the construction of the growth curve. It is necessary to fix the promastigotes in order to count them and this is done by placing them in a Petri dish containing formaldehyde impregnated tissue. This method ensures that they are morphologically unchanged whilst losing their motility.

Table 1. Composition of Medium M199

Reagents Quantity

ddH2O 1 litre

M199 powder (+ HEPES + L-glutamine)

Sigma (M2520) 1 litre units

NaHCO3 2.2 g

Adenosine 13.35 mg

Hemin* 1 mL

Pen/strep** 2 mL

In a clean flask/beaker the M199 powder is added to ~ 800 ml ddH2O. This is mixed well and 2.2 g NaHCO3 is also added. In clean bijoux, the adenosine is dissolved in 5 mL of 10mM NaOH and is then added to the medium by mixing well. The penicillin/streptomycin and hemin aliquots are defrosted and added to the medium. The mediums pH is adjusted to 7.3 - 7.4 and medium is filtered sterilized into 2x 500 mL BD bottles. This is then Stored at 4°C.

Cryopreservation of Leishmania

Approximately 5x106 Leishmania promastigotes are centrifuged (850xg for 10 minutes) and the pellet is then re-suspended in 0.5 mL of the growth medium. The promastigotes are then counted and then placed in cryotube containing growth medium (with 50% FBS and with 10% glycerol) and then the caps are tightly secured. The tubes are frozen gradually by placing into a special "freezing container" called "Mr. Frosty" which has been pre-chilled to refrigerator temperature. The "Mr. Frosty" freezing container (Nalgene) is satisfactory for nearly all Leishmania and other cells. It is inexpensive to purchase and holds eighteen 2-ml cryovials simultaneously. Its contents cool at slightly less than 1oC per 5 minutes when it is placed into a -200C freezer. The freezing container is then placed into a -20oC freezer for 2 hours. Then the cryo-vial are quickly removed from the freezing container, placed into a storage container, and plunged into liquid nitrogen at (-196oC) for indefinite storage.