Next Article in Journal
Exploring Metarhabditis blumi as a Model for Anthelmintic Drug Discovery
Previous Article in Journal
Non-Contact-Lens-Related Acanthamoeba Keratitis Caused by Acanthamoeba sp. Group T4D/T4e
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Parasitologia
Volume 4
Issue 4
10.3390/parasitologia4040027
parasitologia-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

An Overview of Leishmania In Vitro Cultivation and Implications for Antileishmanial Screenings against Promastigotes

by
Virlânio Alves de Oliveira Filho
†,
Marcus Sávio Araujo Garcia
†,
Leticia Bazilio Rosa
,
Selma Giorgio
and
Danilo Ciccone Miguel
*
Programa de Pós-Graduação em Biologia Animal, Instituto de Biologia, Rua Monteiro Lobato, 255, Universidade Estadual de Campinas—UNICAMP, Campinas 13083-862, São Paulo, Brazil
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Parasitologia 2024, 4(4), 305-318; https://doi.org/10.3390/parasitologia4040027
Submission received: 24 July 2024 / Revised: 5 September 2024 / Accepted: 20 September 2024 / Published: 2 October 2024
Versions Notes

Abstract

:
The quest for new drug candidates targeting neglected parasitic diseases has become increasingly urgent over the past decades. Advancements in formulating and optimizing drug delivery systems begin with basic research, including direct assays to evaluate the activity of molecules against parasitic stages maintained in laboratories; i.e., promastigotes. In the context of leishmaniasis, an endemic disease worldwide, the cultivation of Leishmania parasites can vary significantly across different laboratories. Factors such as culture media composition, pH, supplementation, and temperature can lead to varied drug responses in in vitro activity assays. This study aims to compile the parameters used in Leishmania spp. promastigotes cultivation protocols described in scientific articles published in indexed journals over the past ten years. The data reveal a lack of uniformity among Leishmania culture protocols, suggesting a potential bottleneck in comparing the leishmanicidal potential of in vitro drug candidates reported by different research groups. This condition is crucial to consider, because viability/inhibition assays should begin with fully-grown, healthy promastigote cultures capable of homogeneous division, thereby producing more reproducible results.

1. Introduction

1.1. Leishmaniasis

Leishmaniasis belongs to a complex group of parasitic diseases caused by at least 20 species of the Leishmania genus (family Trypanosomatidae), a polymorphic protozoan transmitted by the bite of female sandflies, mainly Phlebotomus and Lutzomyia [1]. Clinical manifestations vary according to geographic location and circulating species, in addition to the immune status of the host, ranging from self-healing skin lesions to fatal visceralization [2].
Cutaneous leishmaniasis is the most prevalent among leishmaniasis, usually causing an ulcerative lesion, except for 10% of cases that progress to more severe complications, such as mucocutaneous leishmaniasis [2,3]. It is estimated that 600,000 to 1 million new cases occur annually in the Americas, Mediterranean countries, the Middle East, and Asia. The visceral form of the disease is characterized by parasite visceralization and hepatosplenomegaly and affects 50,000 to 90,000 new patients every year showing a high potential for outbreak and mortality, according to the World Health Organization [4].
Leishmaniasis chemotherapy relies on a limited array of options, primarily using pentavalent antimonials, Amphotericin B and miltefosine in several countries. These drugs can cause severe side effects and require parenteral administration (except for miltefosine). Consequently, there is an urgent need for alternative treatments, and new drugs are currently being investigated to address these issues [5].

1.2. Parasite Stages and In Vitro Cultivation

Leishmania transmission requires a sandfly infected with promastigotes, flagellate, motile, and extracellular forms, which are regurgitated into the dermis of the mammalian host during a blood meal. The parasites are phagocytosed by macrophages and, within the parasitophorous vacuoles, differentiate and proliferate as amastigotes, an ovoid intracellular form. Ingestion of parasitized cells and free amastigotes by sandflies allows the colonization of freshly differentiated promastigotes in the insect’s gut [6].
The maintenance of Leishmania parasites in vitro is a highly intricate process that involves several variables when considering the complex life cycle that requires distinct culture conditions depending on the parasite’s stage. Cultivating is critical for many different purposes, such as laboratory diagnosis, antigens and antibodies production, drug screenings, molecular characterization of clinical isolates, vaccine tests, in addition to maintaining and storing parasite samples for investigating host–parasite interactions and epidemiological settings [7].
In terms of drug screening, identifying compounds with leishmanicidal activity is a critical step. This involves evaluating various chemical entities for their potential to inhibit or kill Leishmania parasites [8]. Effective drug screening requires robust methodologies, including high-throughput screening (HTS) assays, which enable the rapid testing of thousands of compounds. These assays typically measure the viability, proliferation, or metabolic activity of the parasites upon exposure to potential drugs. Although the use of amastigotes is essential for testing new candidates, the use of promastigotes in leishmanicidal activity assays is still common and well-accepted in the specialized literature [5].
For this reason, the goal of this review is to summarize the cultivation conditions and nutritional requirements for Leishmania promastigote cultivation employed in basic research. In this sense, we expect to shed light on the conditions that are crucial for maintaining the parasites in a consistent and viable state, which is essential for reliable drug screenings. Factors, such as: temperature, pH, and the composition of the culture medium, including the presence of antibiotics, heme sources, specific amino acids, and vitamins are summarized and discussed as key players in the protocols of Leishmania cultivation.

2. Research on Leishmania Cultivation

2.1. Brief Historical Overview of Leishmania Cultivation

The history of leishmaniasis dates back to antiquity, but its etiological agents were described in detail only in the late 19th century. In 1885, Cunningham identified peculiar parasitic organisms in tissue samples in 1885 [9]. Later, in 1898, Brodsky recognized the oval bodies in Oriental sore lesions as protozoa, a finding confirmed by William Boog Leishman and Charles Donovan in 1903 when they identified these bodies in spleen samples from Indian patients [10]. Ronald Ross subsequently classified these as belonging to a new genus, naming them Leishmania donovani [11]. In 1904, Leonard Rogers was the first to successfully cultivate Cunningham–Leishman–Donovan bodies. By adapting the cultivation methods used for trypanosomes, he observed that incubating infected splenic blood at 37 °C led to complete parasite degradation within 24 h. However, at 27 °C, the ovoid bodies multiplied over several days. The third attempt at 22 °C resulted in optimal cultivation conditions, allowing him to observe the emergence of a flagellated form, which he concluded was part of the parasite’s life cycle [12].
The need to isolate, maintain, and grow Leishmania parasites has led to the development and modification of various culture media to improve cultivation efficiency. Today, several commercial media with defined compositions are available, though they may not be accessible to researchers in underdeveloped countries. These media are typically used as base media for additional supplementation based on specific parasite culture needs. For example, the MEM (Minimum Essential Medium) medium is designed for serum supplementation and contains essential inorganic salts, sugars, amino acids, and vitamins, while Medium 199 has a more complete composition and often does not require serum supplementation [13].

2.2. Media for Leishmania Cultivation

The culture medium is crucial in cell cultivation, providing the necessary support for cell survival, proliferation, and function, thus directly affecting study outcomes. Selecting the appropriate culture medium is essential, as understanding its properties helps identify experimental issues [13].
Leishmania cultivation media are classified as semi-solid or biphasic and liquid. Semi-solid media often require blood from rabbit for parasite reproduction, and various additives like brain and heart infusion have been used to enrich the liquid phase. Most liquid media need supplementation with fetal bovine serum (FBS) or rabbit erythrocyte lysate [14]. The media can be serum-supplemented, with undefined composition due to batch-to-batch variation, or serum-free, with well-defined, sometimes protein-free, and chemically defined compositions [15].
One of the first media used for Leishmania in vitro cultivation was a modified version of the Novy–McNeal medium (1904) by Nicolle (1908), known as NNN medium (Novy–McNeal–Nicolle medium). Widely used for diagnosing and isolating Leishmania promastigotes, NNN medium consists of a slanted rabbit blood agar base with an overlaying saline solution, which can range from simple saline to a complete defined medium [16]. The biphasic medium allows Leishmania to thrive, with the solid phase providing essential nutrients [17].
Another common biphasic medium is Evans’ modified Tobie’s medium (EMTM), used for isolating parasites from blood or tissue samples. Like NNN medium, EMTM requires defibrinated rabbit blood in its solid base, along with a liquid phase supplemented with fetal bovine serum (FBS) and human urine [18,19].
The NNN medium, despite its widespread use, presents challenges for lab diagnostics due to the composition variability from body fluids, affecting parasite proliferation, biology and development [20]. Various media using body fluids were developed from the 1910s to the 1950s [20,21,22]. Recognizing the complexity and limitations of these media for functional studies, Morgan and colleagues developed a synthetic media, the medium 199 (M199). M199 underwent extensive optimization, incorporating various combinations of vitamins, amino acids, and other factors to create a defined medium suitable for cell growth. However, it was found that biological samples growth was limited over time without additional supplementation. Consequently, the addition of 5% serum became necessary to support sustained growth [23].

2.3. What Has Been Used for Leishmania Cultivation So Far?

Table 1 demonstrates the diversity of Leishmania species cultivated in both commercial and laboratory-prepared media, alongside various supplements. Selecting the appropriate medium and supplements is a mandatory step that should be considered based on the purpose of promastigote cultivation.
Understanding the doubling time of promastigotes of different strains/species is essential for assays that require logarithmic phase parasites, as a higher number of cells in reduced periods of time may lead to certain nutrient depletion, affecting parasite viability and metacyclogenesis. For example, tests with L. martiniquensis promastigotes revealed that both M199 and Grace media yielded the highest cell counts and shortest doubling times, thus shortening metacyclogenesis phases. Conversely, RPMI 1640 and Schneider’s media had lower yields and longer doubling times, extending the time between metacyclogenesis phases [26]. Conversely, Habibzadeh et al. found that there may be no difference in cell growth, lesion size and parasite load in in vivo assays [25].

2.4. Required Supplementation

Table 2 presents the primary alternatives for replacing FBS traditionally used in culture media, along with substitutes for blood and urine. While the goal is to identify an ideal candidate to eliminate the use of FBS, most studies focus on serum from other animals, which does not address the ethical concerns associated with FBS (Table 2).

2.4.1. Serum

For maintaining and cultivating Leishmania parasites, most culture media are supplemented with FBS, also known as fetal calf serum (FCS). FBS is highly favored due to its rich content of growth-promoting factors, proving crucial for mass culturing of various Leishmania species and strains [43]. It is important to highlight that heat inactivation of serum is crucial for creating a favorable and controlled environment for Leishmania promastigotes. This process deactivates immune components—because only non-dividing metacyclics and amastigotes can survive in the presence of complement system elements—and ensures consistency in serum composition. Additionally, the use of dialyzed serum has also been employed by some research groups, with its primary advantage being the more stringent control of formulation components, despite its higher cost.
Commercial production of FBS involves the use of approximately one million bovine fetuses to yield about 500,000 L of serum worldwide each year, making it a key component for protein supplementation, purine bases, fixation factors, trace elements, growth factors, protective elements, and vitamins. The quality of FBS must be carefully controlled to prevent the spread of zoonoses like Mycoplasma infections, brucellosis, and prion diseases, ensuring a pathogen-free final product [24,35,44]. Despite its widespread use, FBS has several disadvantages, including high cost, batch-to-batch quality variation, composition variability affecting growth and productivity, contamination risks, potential toxicity at high concentrations, and ethical concerns [42,45]. Additionally, FBS is not produced in many countries, particularly in the developing regions where leishmaniasis is most prevalent and much of the research occurs [42].
Given these issues, it is crucial to evaluate the impact of serum on protozoan biology. Consequently, various studies have explored cost-effective and easy-to-produce FBS alternatives for supplementing culture media. Promising substitutes include bovine albumin fraction [46], human plasma [36], chicken serum [39], egg yolk [47], urine [41], milk from cows, buffaloes, and goats [48], and human autologous serum [49].
The search for a defined culture medium without supplementation is crucial due to the high costs and batch-to-batch variability of supplements. Nasiri et al. developed a low-cost LPM (Lithium Phenylethanol Moxalactam) medium that supported L. major promastigote growth similarly to RPMI supplemented with 10% FBS, indicating it could serve as a replacement option. For L. infantum, RPMI (Roswell Park Memorial Institute) 1640 showed the best growth among conventional media (EMTM (Evans’ modified Tobie’s medium) and PY (Peptone Yeast)), but LPM variants RPMI-PY and Tobie-PY, both supplemented with 10% FBS, outperformed RPMI 1640 [27].
Despite these emerging alternatives, FBS remains the primary supplement for Leishmania culture media due to its proven efficacy across various species and strains, even though serum-free methods are being developed.

2.4.2. Blood/Hemin Source

The use of blood is now largely restricted to the NNN medium. The composition of NNN medium typically includes a pancreatic digest of casein, beef liver infusion, and sodium chloride, supplemented with 10% rabbit blood [50]. The medium has a slightly acidic pH of 6.2 to 6.4. Rabbit blood provides essential nutrients for Leishmania parasite growth, while the low pH helps suppress contaminating bacteria. To diagnose leishmaniasis, a sample of the patient’s biological material, such as a skin biopsy or bone marrow aspirate, is inoculated onto the NNN medium and incubated at 25–28 °C for several days. The parasites are visualized microscopically by staining the culture with Giemsa stain.
The NNN medium is a simple and cost-effective culture medium for diagnosing leishmaniasis but has limitations, including slow parasite growth and the need for experienced personnel for culture and microscopy. New diagnostic methods, such as PCR-based tests, may provide faster and more sensitive results. The blood is essential in biphasic and semi-solid culture media like NNN, typically using rabbit or sheep blood, though rabbit blood can be difficult to obtain, and sheep blood is not effective for isolating all Leishmania species. The liquid media often include FBS and other supplements, such as meat extract and peptone [15,27,50].
To substitute blood and the provision of the prosthetic group heme in medium culture, necessary for the metabolism of axenic parasites, researchers take advantage of hemin, an extremely relevant source of heme for the growth of Leishmania, as explored below.
Iron is a biologically and nutritionally significant metal for many microorganisms, playing a crucial role in cellular metabolism stages and being essential for parasite survival, including Leishmania [51]. Heme, a prosthetic group containing an iron atom within a protoporphyrin IX ring, is critical for protein functions such as redox homeostasis, iron acquisition, and oxidation [52]. In eukaryotes, heme biosynthesis involves eight steps, but trypanosomatids lack the final three enzymes needed for protoporphyrin IX ring synthesis [53].
Leishmania parasites are heme auxotrophs, requiring an exogenous heme source. In vivo, Leishmania obtains heme from the host through clathrin-mediated hemoglobin endocytosis [54] and heme salvage with direct transmembrane transport [55,56]. Chang and Chang demonstrated that Leishmania species cultured in vitro depend on an exogenous heme supply, particularly in the form of blood or hemin, for growth, replication, survival, and differentiation of promastigotes and amastigotes in axenic cultures [57]. Surprisingly, many studies do not describe the heme source in the media aliquots used for maintaining Leishmania strains. Trace elements from serum may possibly provide this supply in cultures maintained short to medium-term. Nevertheless, hemin, when included in the media composition, is typically added in a solution containing triethanolamine.

2.4.3. Urine

In recent years, exploring alternative supplementation approaches for Leishmania promastigote cultivation has gained significant attention, particularly as researchers seek cost-effective and readily available options. Among the intriguing alternatives, urine has emerged as a potential source of nutrients and growth factors for these parasites. Urine, often regarded as a waste product, contains a complex mixture of compounds that could potentially support the metabolic needs of Leishmania promastigotes. Studies have shown that urine contains various amino acids, vitamins, and trace elements that are essential for the growth and proliferation of Leishmania parasites, including hydroxybutyrate, acetate, lactate, alanine, and succinate. Allahverdiyev et al. demonstrated the viability of using human urine as a nutrient source for Leishmania promastigotes, showcasing the adaptability of these parasites to utilize unconventional resources for their nutritional requirements. This innovative approach not only sheds light on the adaptable nature of Leishmania but also offers a more sustainable and cost-efficient option for in vitro cultivation [41].
Nasiri et al. observed that adding 2.5% and 5% filtered rabbit or mouse urine to RPMI 1640 medium resulted in significant growth when supplemented with 10% FBS. However, when rabbit or mouse urine at the same concentrations was autoclaved, the growth of L. major promastigotes was lower. By autoclaving the urine, which is rich in amino acids and vitamins, important components for its cycle were degraded, leaving the supplement useless [30].
The use of urine, while intriguing, is not without its challenges. Variability in urine composition due to individual differences and physiological conditions could impact the growth of parasites. Additionally, the optimization of urine-based culture media and the identification of key components responsible for promoting parasite growth warrant further investigation. Nonetheless, the successful utilization of urine as a supplement opens new avenues for designing culture media that rely on locally available and easily accessible resources, particularly in regions where traditional serum supplementation might be limited.

2.4.4. Additional Supplements: Amino Acids, Vitamins and Biopterin

Leishmania utilizes various metabolic pathways to synthesize or acquire nutrients and has numerous peptidases with diverse biological functions, including supplying amino acids [58]. These amino acids are essential for protein synthesis, energy metabolism, differentiation, and osmoregulation [59,60], making them crucial for the parasite’s maintenance.
While Leishmania can biosynthesize many amino acids, some are acquired from the host, due to the absence of certain enzymes required for their production [61,62]. Culture media such as M199 and Schneider’s include various amino acids, though their specific compositions can vary by brand, batch, and date. Commonly included amino acids are alanine, arginine, aspartic acid, cysteine (and its dimer cystine), glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline (and its derivative hydroxyproline), serine, threonine, tryptophan, tyrosine, and valine [63,64]. Regarding L-glutamine, it is relevant to point out that this amino acid is known to be labile, meaning it is sensitive to environmental conditions and can degrade over time. In cell culture, it is especially prone to decomposition when exposed to heat, light, or prolonged storage, which can reduce its effectiveness as a nutrient. Therefore, even commercial media containing L-glutamine often require supplementation during protocol preparation to ensure adequate nutrient levels given its known essentiality for Leishmania growth [65].
The NNN medium, which contains defibrinated rabbit blood, naturally includes amino acids as it is composed of cells, plasma, and other substances [66]. The presence of specific amino acids such as tryptophan, phenylalanine, arginine, leucine, lysine, valine, serine, histidine, tyrosine, and threonine has been found essential for the cultivation and survival of Leishmania promastigotes [31,67]. The culture medium must contain these essential amino acids to support all the processes of an obligate intracellular protozoan, ensuring the integrity and proper cultivation of Leishmania.
Vitamins such as nicotinic acid, folic acid, biotin, thiamine, riboflavin, and pantothenate are crucial nutrients for Leishmania development and replication [29,68]. An emphasis on biotin must be given, due to its essentiality as a cofactor for enzymes involved in energy metabolism and fatty acid synthesis [69]. The absence of biotin in the culture medium may lead to poor growth of the microorganisms, limiting the viability of the cultures [70].
MEM has been utilized for cultivating parasites because it contains essential nutrients such as thiamine, riboflavin, pantothenic acid, pyridoxine, cobalamin, and folic acid. However, its variety DMEM and M199 have been demonstrated to provide a more comprehensive nutritional profile for sustaining mammalian and parasite cell growth, respectively. While the literature suggests that DMEM may be more effective than MEM for mammalian cells [71], studies specifically comparing their potential for cultivating Leishmania are still lacking.
Folic acid and related pteridines are essential cofactors in all life forms, playing crucial roles in major metabolic interconversions involving one-carbon (C1) transfer reactions, including the biosynthesis of thymidylate, purine nucleotides and amino acids [72,73].
Regarding pteridines, it is known that they are essential for the growth of kinetoplastids, including Leishmania [74], and the importance and activity of pterines, including biopterin, was demonstrated in the trypanosomatid Crithidia fasciculata, for which it was observed that the presence of these components in culture media reduces the requirement for folates [75]. In fact, Roy et al. (2011) demonstrated the growth-promoting activity of biopterin in different strains of L. donovani and L. major. However, this activity was only observed in cultures with a low number of passages and a slow growth time. In culture-adapted parasites, biopterin no longer stimulates growth, while folates were not effective in stimulating cell growth even at low passages when compared to biopterin, but it is worth noting that culture media such as SDM-79 are already rich in folates, and M199 supplemented with serum is rich enough, so additional folates are not sufficient for Leishmania growth [72].
In this case, biopterin can be added to the culture if issues with parasite growth arise. Furthermore, the presence of biopterin in mammalian urine may explain its use as a supplement and growth factor for some Old and New World Leishmania species [72].

2.4.5. Antibiotics

In Leishmania promastigote cultivation, antibiotics are commonly employed as a preventive measure against bacterial contamination, which can compromise the integrity of the culture and hinder research outcomes. Although Leishmania parasites themselves are not susceptible to antibiotics like penicillin, streptomycin, and gentamicin, these antibiotics are effective against a wide range of bacteria commonly found in laboratory environments [76]. By incorporating antibiotics into the culture medium, researchers can inhibit bacterial growth and maintain a sterile proper environment to Leishmania proliferation. However, it is mandatory to strike a balance in antibiotic concentrations to prevent interference with the parasites’ viability and growth. Regular monitoring of cultures for signs of contamination and adjustments to antibiotic concentrations as needed are essential practices to ensure the success of Leishmania cultivation and maintain the purity of experimental samples. Overall, judicious use of antibiotics is integral to the establishment and maintenance of healthy and productive Leishmania cultures in laboratory settings.

2.4.6. pH and Temperature

The in vitro cultivation of Leishmania promastigote parasites is intricately influenced by both pH and temperature, critical factors that impact the overall success of the culture. The pH of the culture medium plays a pivotal role in the growth and viability of Leishmania promastigotes. These parasites typically thrive in a slightly acidic to neutral pH range, approximately 7.0 to 7.4. Maintaining this pH range is crucial for providing an environment conducive to Leishmania growth, as deviations can lead to altered parasite morphology, increased protein expression, reduced proliferation, and even cell death [77]. Interestingly, the literature often does not specify the pH values of the culture media used (“NI”, Table 1 and Table 2). Typically, the most common pH values reported are 7, 7.2, and 7.4, with a study reporting the use of several culture media at pH 6.7 [26].
Additionally, Leishmania is highly sensitive to the temperature. The optimal temperature for Leishmania cultivation typically falls within the mesophilic range (25–28 °C) (Table 1 and Table 2), as promastigotes are adapted during infections in sand flies. In the first successful cultivation of the Cunningham–Leishman–Donovan bodies, Leonard Rogers identified that a temperature of 37 °C completely degraded the parasites, while observing that at 27 and 24 °C, there was growth and the appearance of flagella [12].
The temperature influences various cellular processes in Leishmania, including enzymatic activities, metabolic pathways, and the overall rate of replication. Fluctuations outside the optimal range may compromise the parasites’ ability to survive and replicate, impacting the consistency and success of the in vitro culture. In tests to identify the expression of proteins in promastigotes exposed to heat stress, Mule et al. (2024) showed that heat shock-related proteins, proteins involved in metabolism, and ribosomal proteins were most abundantly expressed during the exponential stage in Leishmania spp. [78].
Therefore, meticulous control and monitoring of both pH and temperature are essential for maintaining a stable and supportive environment that promotes the health and growth of Leishmania in laboratory settings.

3. Methods

A systematic review was performed using the PubMed and Scopus databases, restricting results to English-language publications with full-text availability. The keywords “Leishmania”, “culture medium”, and “Leishmania culture” were used to identify twenty studies from the last decade. The review focused on comparing different culture media, introduction of new media and highlighting the significance of various supplements in Leishmania promastigote cultivation. Full-text access was essential for detailed analysis, including parasitic strains, medium components, cultivation conditions, results, and conclusions. Studies published in other languages, those with only abstracts, and those older than eleven years were excluded from our review.

4. Perspectives

Given the heterogeneity of protocols in the cultivation of Leishmania promastigote stages, which are so relevant for the screening of new compounds, a standardization of interlaboratory protocols is suggested. However, the disparity in financial resources between research groups from different countries may be an impediment to the consolidation of standardized methods for the cultivation of these parasites. In an attempt to overcome possible obstacles, the following considerations are highlighted when cultivating Leishmania promastigotes:
-
Preferably use the same batch of M199 or RPMI-1640 from the same supplier;
-
Consider pH range of 7.3–7.4 throughout cultivation;
-
Determine the ideal percentage of FBS (ranging from 10–20%) to be supplemented in culture for testing new compounds, because certain molecules can interact with components of the serum, thus altering its effectiveness;
-
Incubate promastigotes at 24–26 °C in medium supplemented with 5% penicillin/streptomycin, 0.1% hemin (25 mg/mL in 50% triethanolamine), 10 mM adenine (pH 7.5), and 5 mM L-glutamine;
-
Understand the doubling time of Leishmania strains in question and consider the incubation time for leishmanicidal candidates by accounting for the culture’s doubling time and the ratio of drug candidate concentration to the number of parasites per mL;
-
Standardization of the parasite number to obtain homogeneous cells in log and stationary phases.

5. Concluding Remarks

The cultivation of Leishmania has evolved significantly since the early 20th century. The initial efforts using simple media often failed to sustain long-term parasite growth. Over the years, researchers developed more sophisticated culture media, incorporating various nutrients and supplements to better mimic the parasite’s natural environment. These advancements have resulted in more reliable and reproducible cultivation methods, essential for research and drug development. Today, axenic culture techniques enable the growth of different Leishmania species under controlled laboratory conditions, facilitating studies on parasite biology, pathogenicity, and treatment responses. However, standardized protocols should be implemented in laboratory routines to ensure the proper establishment of cell cultivation and supplementation, allowing for comparable results, especially concerning parasite sensitivity to new antileishmanial compounds.

Author Contributions

V.A.d.O.F. and M.S.A.G. have equally contributed to the conceptualization, writing, and preparation of this manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The research received external funding from CNPq (Productivity fellowship PQ#314811/2021-4), CAPES-PRINT (88887.468349/2019-00), and FAPESP (#2014/21129-4). V.A.O.F. and L.B.R. are supported by CNPq Ph.D. fellowships (#141607/2023-8 and #141096/2021-7, respectively) and M.S.G.A. is supported by a CAPES Ph.D. fellowship (#88887.7713008/2022-00). S.G. thanks to CNPq for Productivity Fellowship (PQ#30439/2021-4).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Torres-Guerrero, E.; Quintanilla-Cedillo, M.R.; Ruiz-Esmenjaud, J.; Arenas, R. Leishmaniasis: A Review. F1000Research 2017, 6, 750. [Google Scholar] [CrossRef]
  2. Burza, S.; Croft, S.L.; Boelaert, M. Leishmaniasis. Lancet 2018, 392, 951–970. [Google Scholar] [CrossRef]
  3. Sabzevari, S.; Mohebali, M.; Hashemi, A. Cutaneous and Visceral Leishmaniasis: Parasites, Vectors and Reservoir Hosts in Endemic Foci of North Khorasan, Northeastern Iran—A Narrative Review. J. Med. Microbiol. Infect. Dis. 2020, 8, 40–44. [Google Scholar] [CrossRef]
  4. World Health Organization. Leishmaniasis. 2023. Available online: https://www.who.int/health-topics/leishmaniasis#tab=tab_1 (accessed on 28 June 2024).
  5. Alcântara, L.M.; Ferreira, T.C.; Gadelha, F.R.; Miguel, D.C. Challenges in Drug Discovery Targeting TriTryp Diseases with an Emphasis on Leishmaniasis. Int. J. Parasitol. Drugs Drug Resist. 2018, 8, 430–439. [Google Scholar] [CrossRef]
  6. Serafim, T.D.; Coutinho-Abreu, I.V.; Dey, R.; Kissinger, R.; Valenzuela, J.G.; Oliveira, F. Shaden Kamhawi Leishmaniasis: The Act of Transmission. Trends Parasitol. 2021, 37, 976–987. [Google Scholar] [CrossRef]
  7. Visvesvara, G.S.; Garcia, L.S. Culture of Protozoan Parasites. Clin. Microbiol. Rev. 2002, 15, 327–328. [Google Scholar] [CrossRef]
  8. Sereno, D.; Silva, A.C.; Mathieu-Daude, F.; Ouaissi, A. Advances and Perspectives in Leishmania Cell Based Drug-Screening Procedures. Parasitol. Int. 2007, 56, 3–7. [Google Scholar] [CrossRef]
  9. Steverding, D. The History of Leishmaniasis. Parasites Vectors 2017, 10, 82. [Google Scholar] [CrossRef]
  10. Leishman, W.B. On the possibility of the occurrence of trypanosomiasis in India. Br. Med. J. 1903, 1, 1252–1254. [Google Scholar] [CrossRef]
  11. Ross, R. Note on the bodies recently described by Leishman and Donovan. Br. Med. J. 1903, 2, 1261. [Google Scholar] [CrossRef]
  12. Rogers, L. Preliminary note on the development of Trypanosoma in cultures of the Cunningham-Leishman-Donovan bodies of cachexial fever and kala-azar. Lancet 1904, 164, 215–216. [Google Scholar] [CrossRef]
  13. Yao, T.; Asayama, Y. Animal-Cell Culture Media: History, Characteristics, and Current Issues. Reprod. Med. Biol. 2017, 16, 99–117. [Google Scholar] [CrossRef] [PubMed]
  14. Sharief, A.H.; Khalil, E.A.G.A.; Suliaman, S.M. In Vitro Cultivation of Leishmania donovani Promastigotes: Growth Potential of Human Urine as Replacement of Fetal Calf Serum. Ann. Syst. Biol. 2021, 4, 001–004. [Google Scholar] [CrossRef]
  15. Ladopoulos, T.; Ntais, P.; Tsirigotakis, N.; Dokianakis, E.; Antoniou, M. The Proliferation Potential of Promastigotes of the Main Leishmania Species of the Old World in NNN Culture Medium Prepared Using Blood of Four Different Mammals. Exp. Parasitol. 2015, 157, 124–127. [Google Scholar] [CrossRef]
  16. Nicolle, C.H. Culture du parasite du Bouton d’Orient. C. R. Acad. Sci. 1908, 146, 842–843. [Google Scholar]
  17. Hai, N.; Akhter, R.P.; Ali, I. Isolation, Maintenance and Detection of Leishmania Parasite by Microscopy and Culture Technique. In Proceedings of the 2017 14th International Bhurban Conference on Applied Sciences and Technology (IBCAST), Islamabad, Pakistan, 10–14 January 2017. [Google Scholar] [CrossRef]
  18. Evans, D.A.  Leishmania. In In-Vitro Methods for Parasite Cultivation; Taylor, A.E., Baker, J.R., Eds.; Academic Press: London, UK, 1987; pp. 52–75. [Google Scholar]
  19. Tobie, E.J.; von Brand, T.; Mehlman, B.A. Cultural and Physiological Observations on Trypanosoma rhodesiense and Trypanosoma gambiense. J. Parasitol. 1950, 36, 48. [Google Scholar] [CrossRef] [PubMed]
  20. Chang, S.L. Studies on Haemoflagellates: I. A Semi-Solid Medium and a Fluid Medium with a Solid Base for Growing Various Species of Leishmania and Trypanosoma cruzi. J. Infect. Dis. 1947, 80, 164–171. [Google Scholar] [CrossRef]
  21. Salle, A.J.; Schmidt, C.L.A. The metabolism of Leishmania tropica. J. Infect. Dis. 1928, 43, 378–384. [Google Scholar] [CrossRef]
  22. Fulton, J.D.; Joyner, L.P. Studies on Protozoa. Part I. The Metabolism of Leishman-Donovan Bodies and Flagellates of Leishmania donovani. Trans. R. Soc. Trop. Med. Hyg. 1949, 43, 273–286. [Google Scholar] [CrossRef]
  23. Morgan, J.F.; Morton, H.J.; Parker, R.C. Nutrition of Animal Cells in Tissue Culture. I. Initial Studies on a Synthetic Medium. Exp. Biol. Med. 1950, 73, 1–8. [Google Scholar] [CrossRef]
  24. Costa, A.F.P.; Brito, R.C.F.; Carvalho, L.M.; Cardoso, J.M.O.; Vieira, P.M.A.; Reis, A.B.; Soares, R.D.O.A.; Roatt, B.M. Liver infusion tryptose (LIT): The best choice for growth, viability, and infectivity of Leishmania infantum parasites. Parasitol. Res. 2020, 119, 4185–4195. [Google Scholar] [CrossRef] [PubMed]
  25. Habibzadeh, S.; Doroud, D.; Taheri, T.; Seyed, N.; Rafati, S. Leishmania Parasite: The Impact of New Serum-Free Medium as an Alternative for Fetal Bovine Serum. PubMed 2021, 25, 349–358. [Google Scholar] [CrossRef]
  26. Siripattanapipong, S.; Boontanom, P.; Leelayoova, S.; Mungthin, M.; Tan-ariya, P. In Vitro Growth Characteristics and Morphological Differentiation of Leishmania martiniquensis Promastigotes in Different Culture Media. Acta Trop. 2019, 197, 105039. [Google Scholar] [CrossRef] [PubMed]
  27. Nasiri, V.; Dalimi, A.; Ghaffarifar, F. LB Broth-Lyophilized Rabbit Serum (LLR) as a New and Suitable Culture Medium for Cultivation of Promastigotes of Leishmania major. J. Parasit. Dis. 2017, 41, 247–251. [Google Scholar] [CrossRef]
  28. Castelli, G.; Galante, A.; Verde, V.L.; Migliazzo, A.; Reale, S.; Lupo, T.; Piazza, M.; Vitale, F.; Bruno, F. Evaluation of two modified culture media for Leishmania infantum cultivation versus different culture media. J. Parasitol. 2014, 100, 228–230. [Google Scholar] [CrossRef]
  29. Santarém, N.; Cunha, J.; Silvestre, R.; Silva, C.; Moreira, D.; Ouellette, M.; Cordeiro-Da-Silva, A. The Impact of Distinct Culture Media in Leishmania Infantum Biology and Infectivity. Parasitology 2013, 141, 192–205. [Google Scholar] [CrossRef]
  30. Nasiri, V. Sheep Blood–LB Agar Base Medium (SLM) as a Simple and Suitable Medium for the Cultivation of Leishmania major Promastigotes. Parasitol. Res. 2013, 112, 3741–3742. [Google Scholar] [CrossRef]
  31. Nayak, A.; Akpunarlieva, S.; Barrett, M.; Burchmore, R. A Defined Medium for Leishmania Culture Allows Definition of Essential Amino Acids. Exp. Parasitol. 2018, 185, 39–52. [Google Scholar] [CrossRef]
  32. Grekov, I.; Svobodová, M.; Nohýnková, E.; Lipoldová, M. Preparation of Highly Infective Leishmania Promastigotes by Cultivation on SNB-9 Biphasic Medium. J. Microbiol. Methods 2011, 87, 273–277. [Google Scholar] [CrossRef]
  33. Abhishek, S.; Chaturvedi, S.K.; Gupta, A.K.; Ahmed, G.; Das, V.R.; Narayan, S. A Simple Monophasic LGPY Medium for Routine Maintenance of Leishmania donovani Promastigotes. Parasitol. Res. 2021, 120, 2969–2971. [Google Scholar] [CrossRef]
  34. Rahi, A.A. Cutaneous Leishmaniasis in Iraq: A Clinicoepidemiological Descriptive Study. Sch. J. Appl. Med. Sci. (SJAMS) 2013, 1, 1021–1025. [Google Scholar] [CrossRef]
  35. Esfandiari, F.; Derakhshanfar, A.; Goudarzi, F.; Hatam, G. Comparison of Camel, Dog and the Laboratory Animals’ Sera with the Fetal Calf Serum (FCS) for Cultivation of Leishmania major. J. Parasit. Dis. 2020, 44, 299–304. [Google Scholar] [CrossRef] [PubMed]
  36. Mahamoud, A.; Osman, H.A.; Mansour, D.; el Harith, A. Successful Substitution of Fetal Calf Serum by Human Plasma for Bulk Cultivation of Leishmania donovani Promastigotes. J. Med. Microbiol. 2013, 62, 1165–1169. [Google Scholar] [CrossRef] [PubMed]
  37. Nasiri, V.; Karimi, G.; Dalimi, A.; Paykari, H.; Ghaffarifar, F. Effects of Sheep and Mouse Urine on the Growth Pattern Of Leishmania major Promastigotes. BioMed Res. Int. 2013, 2013, 748592. [Google Scholar] [CrossRef]
  38. Rahmani, Z.; Faridnia, R.; Kalani, H.; Ghanei, N.; Fakhar, M.; Zamanian, M.; Keighobadi, M.; Tabaripour, R. Comparative Evaluation of Amniotic Fluid as an Alternative to Fetal Bovine Serum in the Maintenance of Leishmania major and Toxoplasma gondii. Parasitol. Res. 2021, 120, 1059–1065. [Google Scholar] [CrossRef] [PubMed]
  39. Nasiri, V.; Dalimi, A.; Ghaffarifar, F. Use of Chicken (Gallus gallus) Serum as a Costly Replacement for the Fetal Calf Serum in Cultivation of Promastigotes of Leishmania infantum. Asian Pac. J. Trop. Dis. 2013, 3, 169–173. [Google Scholar] [CrossRef]
  40. Santos, R.C.; Pinho, F.A.; Passos, G.P.; Larangeira, D.F.; Barrouin-Melo, S.M. Isolation of Naturally Infecting Leishmania infantum from Canine Samples in Novy-MacNeal-Nicolle Medium Prepared with Defibrinated Blood from Different Animal Species. Vet. Parasitol. 2018, 257, 10–14. [Google Scholar] [CrossRef]
  41. Allahverdiyev, A.M.; Bagirova, M.; Elcicek, S.; Koc, R.C.; Oztel, O.N. Effect of Human Urine on Cell Cycle and Infectivity of Leismania Species Promastigotes In Vitro. Am. J. Trop. Med. Hyg. 2011, 85, 639–643. [Google Scholar] [CrossRef] [PubMed]
  42. Nasiri, V.; Habibi, G.; Dalimi, A.; Esmailnia, K. Use of Chicken Serum as a Good Replacement for the Fetal Calf Serum in Cultivation of Promastigotes of Leishmania major. Arch. Razi Inst. 2011, 66, 59–64. [Google Scholar] [CrossRef]
  43. Lemesre, J.L.; Darcy, F.; Kweider, M.; Capron, A.; Santoro, F. Requirements of defined cultivation conditions for standard growth of Leishmania promastigotes in vitro. Acta Trop. 1988, 45, 99–108. [Google Scholar]
  44. van der Valk, J.; Mellor, D.; Brands, R.; Fischer, R.; Gruber, F.; Gstraunthaler, G.; Hellebrekers, L.; Hyllner, J.; Jonker, F.H.; Prieto, P.; et al. The Humane Collection of Fetal Bovine Serum and Possibilities for Serum-Free Cell and Tissue Culture. Toxicol. Vitr. 2004, 18, 1–12. [Google Scholar] [CrossRef] [PubMed]
  45. van der Valk, J.; Bieback, K.; Buta, C.; Cochrane, B.; Dirks, W.G.; Fu, J.; Hickman, J.J.; Hohensee, C.; Kolar, R. Fetal bovine serum (FBS): Past–present–future. Altex 2018, 35, 1–20. [Google Scholar] [CrossRef] [PubMed]
  46. Sunter, J.; Gull, K. Shape, form, function and Leishmania pathogenicity: From textbook descriptions to biological understanding. Open Biol. 2017, 7, 170165. [Google Scholar] [CrossRef] [PubMed]
  47. Sasse, M.; Lengwinat, T.; Henklein, P.; Hlinak, A.; Schade, R. Replacement of fetal calf serum in cell cultures by an egg yolk factor with cholecystokinin/gastrin-like immunoreactivity. Altern. Lab. Anim. 2000, 28, 815–831. [Google Scholar] [CrossRef]
  48. Muniaraj, M.; Lal, C.S.; Kumar, S.; Sinha, P.K.; Das, P. Milk of Cow (Bos taurus), Buffalo (Bubalus bubalis), and Goat (Capra hircus): A Better Alternative than Fetal Bovine Serum in Media for Primary Isolation, in Vitro Cultivation, and Maintenance of Leishmania donovani Promastigotes. J. Clin. Microbiol. 2007, 45, 1353–1356. [Google Scholar] [CrossRef]
  49. Goodarzi, P.; Arjmand, B.; Emami-Razavi, S.H.; Soleimani, M.; Khodadadi, A.; Mohamadi-Jahani, F.; Aghayan, H.R. Human Autologous Serum as a Substitute for Fetal Bovine Serum in Human Schwann Cell Culture. Acta Medica Iran. 2014, 52, 241–245. [Google Scholar]
  50. Castelli, G.; Oliveri, E.; Valenza, V.; Giardina, S.; Facciponte, F.; Russa, F.L.; Vitale, F.; Bruno, F. Cultivation of Protozoa Parasites in Vitro: Growth Potential in Conventional Culture Media versus RPMI-PY Medium. Vet. Sci. 2023, 10, 252. [Google Scholar] [CrossRef]
  51. Zaidi, A.; Singh, K.P.; Ali, V. Leishmania and Its Quest for Iron: An Update and Overview. Mol. Biochem. Parasitol. 2017, 211, 15–25. [Google Scholar] [CrossRef] [PubMed]
  52. Flannery, A.R.; Renberg, R.L.; Andrews, N.W. Pathways of Iron Acquisition and Utilization in Leishmania. Curr. Opin. Microbiol. 2013, 16, 716–721. [Google Scholar] [CrossRef]
  53. Kořený, L.; Lukeš, J.; Oborník, M. Evolution of the Haem Synthetic Pathway in Kinetoplastid Flagellates: An Essential Pathway That Is Not Essential after All? Int. J. Parasitol. 2010, 40, 149–156. [Google Scholar] [CrossRef]
  54. Agarwal, S.; Rastogi, R.; Gupta, D.; Patel, N.; Raje, M.; Mukhopadhyay, A. Clathrin-Mediated Hemoglobin Endocytosis Is Essential for Survival of Leishmania. Biochim. Et Biophys. Acta. Mol. Cell Res. 2013, 1833, 1065–1077. [Google Scholar] [CrossRef] [PubMed]
  55. Huynh, C.; Yuan, X.; Miguel, D.C.; Renberg, R.L.; Protchenko, O.; Philpott, C.C.; Hamza, I.; Andrews, N.W. Heme Uptake by Leishmania amazonensis Is Mediated by the Transmembrane Protein LHR1. PLoS Pathog. 2012, 8, e1002795. [Google Scholar] [CrossRef] [PubMed]
  56. Miguel, D.C.; Flannery, A.R.; Mittra, B.; Andrews, N.W. Heme Uptake Mediated by LHR1 Is Essential for Leishmania amazonensis Virulence. Infect. Immun. 2013, 81, 3620–3626. [Google Scholar] [CrossRef] [PubMed]
  57. Chang, C.S.; Chang, K.P. Heme Requirement and Acquisition by Extracellular and Intracellular Stages of Leishmania mexicana amazonensis. Mol. Biochem. Parasitol. 1985, 16, 267–276. [Google Scholar] [CrossRef]
  58. Klemba, M.; Gluzman, I.; Goldberg, D.E. A Plasmodium falciparum Dipeptidyl Aminopeptidase I Participates in Vacuolar Hemoglobin Degradation. J. Biol. Chem. 2004, 279, 43000–43007. [Google Scholar] [CrossRef]
  59. Bringaud, F.; Rivière, L. Virginie Coustou Energy Metabolism of Trypanosomatids: Adaptation to Available Carbon Sources. Mol. Biochem. Parasitol. 2006, 149, 1–9. [Google Scholar] [CrossRef]
  60. Silber, A.; Colli, W.; Ulrich, H.; Alves, M.M.; Pereira, C. Amino Acid Metabolic Routes in Trypanosoma cruzi: Possible Therapeutic Targets against Chagas; Disease. Curr. Drug Targets. Infect. Disord. 2005, 5, 53–64. [Google Scholar] [CrossRef]
  61. Vieira, L.L.; Cabantchik, Z.I. Amino Acid Uptake and Intracellular Accumulation in Leishmania major Promastigotes Are Largely Determined by an H+-Pump Generated Membrane Potential. Mol. Biochem. Parasitol. 1995, 75, 15–23. [Google Scholar] [CrossRef]
  62. Payne, S.H.; Loomis, W.F. Retention and Loss of Amino Acid Biosynthetic Pathways Based on Analysis of Whole-Genome Sequences. Eukaryot. Cell 2006, 5, 272–276. [Google Scholar] [CrossRef]
  63. Morgan, J.F.; Campbell, M.E.; Morton, H.J. The nutrition of animal tissues cultivated in vitro. I. A survey of natural materials as supplements to synthetic medium 199. J. Natl. Cancer Inst. 1955, 16, 557–567. [Google Scholar] [CrossRef]
  64. Morton, H.J.; Tolnai, S. Preparation of Medium 199. TCA Man. 1978, 4, 729–736. [Google Scholar] [CrossRef]
  65. Kumar, V.; Ghosh, S.; Roy, K.; Pal, C.; Singh, S. Deletion of Glutamine Synthetase Gene Disrupts the Survivability and Infectivity of Leishmania donovani. Front. Cell. Infect. Microbiol. 2021, 11, 622266. [Google Scholar] [CrossRef]
  66. Hendricks, L.; Wright, N. Diagnosis of Cutaneous Leishmaniasis by in Vitro Cultivation of Saline Aspirates in Schneider’s Drosophila Medium. Am. J. Trop. Med. Hyg. 1979, 28, 962–964. [Google Scholar] [CrossRef] [PubMed]
  67. Trager, W. Nutrition of a Hemoflagellate (Leishmania tarentolae) Having an Interchangeable Requirement for Choline or Pyridoxal. J. Protozool. 1957, 4, 269–276. [Google Scholar] [CrossRef]
  68. Schuster, F.L.; Sullivan, J.J. Cultivation of Clinically Significant Hemoflagellates. Clin. Microbiol. Rev. 2002, 15, 374–389. [Google Scholar] [CrossRef] [PubMed]
  69. Zhao, J.R.; Zuo, S.Q.; Xiao, F.; Guo, F.Z.; Chen, L.Y.; Bi, K.; Cheng, D.Y.; Xu, Z.N. Advances in Biotin Biosynthesis and Biotechnological Production in Microorganisms. World J. Microbiol. Biotechnol. 2024, 40, 163. [Google Scholar] [CrossRef] [PubMed]
  70. Satiaputra, J.; Eijkelkamp, B.A.; McDevitt, C.A.; Shearwin, K.E.; Booker, G.W.; Polyak, S.W. Biotin-Mediated Growth and Gene Expression in Staphylococcus Aureus Is Highly Responsive to Environmental Biotin. Appl. Microbiol. Biotechnol. 2018, 102, 3793–3803. [Google Scholar] [CrossRef]
  71. Riis, S.; Nielsen, F.M.; Pennisi, C.P.; Zachar, V.; Fink, T. Comparative Analysis of Media and Supplements on Initiation and Expansion of Adipose-Derived Stem Cells. Stem Cells Transl. Med. 2016, 5, 314–324. [Google Scholar] [CrossRef]
  72. Roy, G.; Kündig, C.; Olivier, M.; Papadopoulou, B.; Ouellette, M. Adaptation of Leishmania Cells to in Vitro Culture Results in a More Efficient Reduction and Transport of Biopterin. Exp. Parasitol. 2001, 97, 161–168. [Google Scholar] [CrossRef]
  73. Vickers, T.J.; Beverley, S.M. Folate Metabolic Pathways in Leishmania. Essays Biochem. 2011, 51, 63–80. [Google Scholar] [CrossRef]
  74. Trager, W. Pteridine Requirement of the Hemoflagellate Leishmania tarentolae. J. Protozool. 1969, 16, 372–375. [Google Scholar] [CrossRef] [PubMed]
  75. Kindder, G.W.; Dutta, B.N. The Growth and Nutrition of Crithidia Fasciculata. J. Gen. Microbiol. 1958, 18, 621–638. [Google Scholar] [CrossRef] [PubMed]
  76. Hyun, S.-W.; Kim, B.-R.; Lin, D.; Hyun, S.-A.; Yoon, S.S.; Seo, J.-W. The Effects of Gentamicin and Penicillin/Streptomycin on the Electrophysiology of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Manual Patch Clamp and Multi-Electrode Array System. J. Pharmacol. Toxicol. Methods 2018, 91, 1–6. [Google Scholar] [CrossRef] [PubMed]
  77. Gomes, C.B.; Souza-Silva, F.; Charret, K.S.; Pereira, B.A.S.; Finkelstein, L.C.; Santos-de-Souza, R.; Côrtes, L.M.C.; Pereira, M.C.S.; Oliveira, F.O.R., Jr.; Alves, C.R. Increasing in Cysteine Proteinase B Expression and Enzymatic Activity during in Vitro Differentiation of Leishmania (Viannia) Braziliensis: First Evidence of Modulation during Morphological Transition. Biochimie 2017, 133, 28–36. [Google Scholar] [CrossRef]
  78. Mule, S.N.; Saad, J.S.; Sauter, I.P.; Fernandes, L.R.; Oliveira, G.S.; Quina, D.; Tano, F.T.; Brandt-Almeida, D.; Padrón, G.; Stolf, B.S.; et al. The Protein Map of the Protozoan Parasite Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) Infantum during Growth Phase Transition and Temperature Stress. J. Proteom. 2024, 295, 105088. [Google Scholar] [CrossRef]
Table 1. Detailed Leishmania culture media used in laboratory routine over the last decade.
Table 1. Detailed Leishmania culture media used in laboratory routine over the last decade.
Medium (Brand)ComponentsSerumAntibioticspH/Temp. (°C)Leishmania sp. (Strain)Reference
Liver Infusion Tryptose (LIT) (Sigma, Livonia, MI, USA)Four-salt solution (NaCl, KCl, Na2HPO4, tryptose) supplemented with bovine hemin in addition to a 10% liver infusion broth and a 40% glucose solutionNone; 10% faction V bovine albumin; 10% FBS.200 U/mL penicillin; 200 U/mL streptomycinNI/23–24L. infantum (MCAN/BR/08/OP46)[24]
Grace’s (Gibco, Grand Island, NY, USA)@
Schneider’s (Sigma, USA)@
CSFM (Complete Serum-Free defined Medium)MEME as base medium supplemented with sodium bicarbonate, MEM non-essential amino acid solution, MEM amino acids, HEPES, L-glutamine, hemin, BME vitamins solution, and D-glucose- 7.2/26L. tarentolae Tar II (ATCC 30.267)
L. tarentolae-EGFP
L. tarentolae-PpSP15-EGFP
L. major (MRHO/IR/75/ER)		[25]
M199 (Sigma, Steinheim, Germany)M199 supplemented with sodium bicarbonate, adenosine, HEPES, L-glutamine, and hemin5% FBS100 µg/mL gentamicin
M199 (Gibco BRL, Saint Egrève, France)M199 supplemented with L-glutamine, HEPES, phenol red and hemin20% FBSNone6.7/25L. martiniquensis (MHOM/TH/2011/PG)[26]
RPMI-1640 (Gibco BRL, France)RPMI-1640 supplemented with L-glutamine and phenol red
Grace’s (Gibco BRL, France)Grace’s supplemented with L-glutamine
Schneider’s (Sigma, USA)Schneider’s supplemented with L-glutamine
Luria-Bertani (LB) brothPeptone, Yeast Extract and NaCl (for 100 mL).1, 2.5, 5 and 10% lyophilized rabbit serumNoneNI/26L. major (MRHO/IR/76/ER)[27]
RPMI-1640 10% FBS
Evans’ modified Tobie’s medium (EMTM)Solid Tobie (beef extract, bacteriological peptone, NaCl, agar, and defibrinated rabbit blood) with liquid Tobie (KCl, Na2HPO4, KH2PO4, CaCl2, MgSO4, MgCl2 and NaCl) supplemented with L-proline and phenol red10% FBS250 μg/mL gentamicin7.2/24L. infantum (IPT1 ZMON1)[28]
RPMI-1640 (Gibco, USA)RPMI-1640 supplemented with L-glutamine-
Peptone-Yeast extract medium (P-Y)Peptone, yeast extract, Na2HPO4, NaCl and glutamine100 U/mL penicillin; 0.1 mg/mL streptomycin.
RPMI-PYRPMI-1640 and PY (v:v)250 μg/mL gentamicin
Tobie-PYPY and liquid Tobie (v:v)
SDM-79SDM-79 supplemented with hemin and biopterin10% FBS NI/27L. infantum (MHOM/MA/67/ITMAP-263)[29]
RPMI-1640 (Lonza, Basel, Switzerland)RPMI-1640 supplemented with L-glutamine and HEPES.100 U/mL penicillin; 100 mg/mL streptomycin
Schneider’s (Sigma, USA)Schneider’s supplemented with HEPES and phenol red.200 U/mL penicillin; 200 U/mL streptomycin
NNNNaCl, defibrinated rabbit blood and agar; RPMI as liquid phase 625 U/mL penicillin; 625 U/mL streptomycin
SLMLuria-Bertani (LB) agar plus defibrinated sheep blood as the base and LB broth as the liquid phase- NI/26L. major (MRHO/IR/76/ER)[30]
HOMEM (Invitrogen, Carlsbad, CA, USA)@ 10% FBS 7.4/27L. mexicana (MNYC/BZ/62/M379)
L. donovani (MHOM/SD/63/Khartoum)
L. major (MHOM/IL/80/Friedlin)		[31]
Nayak mediumModified composition of RE IX medium to include equimolar concentrations (0.5 mM) of all 20 proteinogenic amino acids
SNB-9Blood agar base (neopeptone, NaCl, rabbit defibrinated blood and agar) with a liquid phase (neopeptone and NaCl solution)10 or 20% FBS50 μg/mL gantamicinNI/23L. major (MHOM/IL/67/LRC-L137 JERICHO II)
L. tropica (MHOM/TR/98/HM)		[32]
Schneider’s (Sigma, USA)Schneider’s supplemented with 2% urine 50 μg/mL gantamicin; 63.7 μg/mL penicillin; 100 μg/mL streptomycin
LGPYLocke’s salts type inorganic mixture (NaCl, KCl, KH2PO4 and CaCl2) enriched with glucose, peptone and yeast extract10% FBS 7.4/24L. donovani (MHOM/IN/80/DD8)[33]
RPMI-1640@
M199@
NNNSolid phase(brain heart infusion, agar, D-Glucose, defibrinated rabbit blood) and a liquid phase (NaCl, KCl, CaCl2, NaHCO3, and glucose 80 mg/mL gantamicinNI/25Leishmania sp. (isolated)[34]
RPMI-1640@10% FBS1 U/100 mL penicillin; 1 gm/100
mL streptomycin
@: Prepared according to the manufacturer’s instructions; FBS: Fetal bovine serum; NI: not informed.
Table 2. Supplementation options employed in culture media for Leishmania cultures.
Table 2. Supplementation options employed in culture media for Leishmania cultures.
Medium (Brand)SupplementationAntibioticspH/Temp. (°C)Leishmania sp. (Strain)Ref.
RPMI-1640 (Sigma)10% FBS; BALB/c; rat; guinea pig; hamster; rabbit; dog; camel; cocktail hamster + guinea pig + BALB serum100 U/mL penicillin; 100 µg/mL streptomycinNI/25L. major (MRHO/IR/75/ER)[35]
NNNDonkey, horse, sheep, and goat blood NI/26 ± 1L. infantum;
L. donovani;
L. tropica;
L. major		[15]
LIT15% FBS; pooled human sera; individual human plasma. 7.4/24–26L. donovani[36]
RPMI-1640 (Sigma)10% FBS (control); 1, 2.5, 5, and 10% urine of sheep or mouse 7/26L. major (MRHO/IR/76/ER)[37]
RPMI-1640 (Gibco)2, 5, 10, 20, and 30% FBS or amniotic fluid NI/24L. major (MRHO/IR/75/ER)[38]
RPMI-1640 (Sigma)10% FBS (control); 1, 2.5, 5, and 10% of chicken serum 7/26L. infantum (MCAN/IR/07/Moheb-gh.)[39]
NNNOvine, bovine, equine, chicken, and rabbit blood NI/24–26L. infantum (isolated)[40]
RPMI-1640 (Sigma)5, 10, 15, 20, 25, and 30% human urine80 μg/mL gentamicinNI/27L. tropica
L. infantum
L. donovani
L. major		[41]
RPMI-1640 (Sigma)10% FBS (control); 1, 2.5, 5, and 10% chicken serumNone7/26L. major (MRHO/IR/75/ER)[42]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

de Oliveira Filho, V.A.; Garcia, M.S.A.; Rosa, L.B.; Giorgio, S.; Miguel, D.C. An Overview of Leishmania In Vitro Cultivation and Implications for Antileishmanial Screenings against Promastigotes. Parasitologia 2024, 4, 305-318. https://doi.org/10.3390/parasitologia4040027

AMA Style

de Oliveira Filho VA, Garcia MSA, Rosa LB, Giorgio S, Miguel DC. An Overview of Leishmania In Vitro Cultivation and Implications for Antileishmanial Screenings against Promastigotes. Parasitologia. 2024; 4(4):305-318. https://doi.org/10.3390/parasitologia4040027

Chicago/Turabian Style

de Oliveira Filho, Virlânio Alves, Marcus Sávio Araujo Garcia, Leticia Bazilio Rosa, Selma Giorgio, and Danilo Ciccone Miguel. 2024. "An Overview of Leishmania In Vitro Cultivation and Implications for Antileishmanial Screenings against Promastigotes" Parasitologia 4, no. 4: 305-318. https://doi.org/10.3390/parasitologia4040027

APA Style

de Oliveira Filho, V. A., Garcia, M. S. A., Rosa, L. B., Giorgio, S., & Miguel, D. C. (2024). An Overview of Leishmania In Vitro Cultivation and Implications for Antileishmanial Screenings against Promastigotes. Parasitologia, 4(4), 305-318. https://doi.org/10.3390/parasitologia4040027

Article Metrics

Back to TopTop