We are planning to start in vitro work with Trypanosoma brucei. But we have been facing some challenges in culturing from cryopreserved vials, using Iscove’s Modified Dulbecco’s Medium (25 mM HEPES-buffered D-MEM+L-Glutamine), supplemented with NaOHCO3, Hypoxanthine, Thymidine, Glucose, Sodium pyruvate, gentamicin and cysteine. Something seems to be going wrong, despite the fact that we are applying the culture conditions published by renown scientists.
I presume you are culturing bloodstream form T.brucei not procyclic forms based on the medium you desrcibe above. I also assume that you are trying to culture T.brucei brucei, not the human infective subspecies and that you are using bovine foetal calf serum. From what you mention above, I do not see the inclusion of mercaptoethanol (or thioglycerol) in your medium or the chelator bathocuprione. These are essential to prevent the oxidation of cysteine to cystine and hydrogen peroxide, While pyruvate prevents toxicity by hydrogen peroxide, trypanosomes are cysteine auxotrophs and incapable of growing on cystine (cysteine disulfide). The problem may also lie in the stabilate, but after thawing you should see evidience of a least some motile trypanosomes, indicative of viability. There are other factors, but Hirumi's HMI9 and HMI11 media and Baltz's medium all should support robust growth. I can think of lots of other possibilities, but that should be enough for you to start with in troubleshooting!
Good luck!
HI Denis,
I don't have much to add Alan's comprehensive answer above, but it would be helpful if you supplied more details. Are you certain of which (sub)-species of trypanosome you are trying to culture? T. b. brucei and T. b. rhodesiense should be possible in the media Alan mentions, but T. b. gambiense is very difficult to bring into culture, as are T. congolense and T. vivax. Are you bringing the trypanosomes into the culture by taking them directly from frozen stabilates (if so, have you checked microscopically that they are viable?) or from an infected animal? In the latter case, did you first purify the parasites, or add some infected blood directly into the culture medium? Are you using antibiotics to keep the culture free of bacterial contamination? We prefer not to do so, as it can potentially impact on the trypanosomes as well, depending on the type of antibiotic and the concentration.
All the best,
Harry de Koning
Dear Alan and Harry,
Thanks so much for your contributions and assistance. We are planning to culture both T. b. brucei, T b gambiense and T. b. Lister. But we started with T b Lister and T. b brucei. That is where we faced the challenge. We are culturing from cryopreserved vials. Of course we checked and confirmed the viability and high motility under microscope. The following day, parasites have undergone shape modification, and the flagella were no more. They remained like that for days before disappearing. We were using gentamycin (50 ug/ml) to prevent bacteria contamination. Obviously something went wrong, like a kind of toxicity against the parasites. That's what we struggling to understand and correct.
Hi everyone!, we have seen a similar problem with in vitro free-layer culture of Trypanosoma evansi strains, coinciding with De Koning´s comment about T. b. gambiense, so that these behaviours could be correlated with Cuypers et al (2017) classification of Trypanozoon species according SNP phylogenetic analysis.
Hi, check this paper: Article Standard culture medium allows clonal dilution of Trypanosom...
, It may help you.
May I ask a question too...
please, i've been struggling to separate trypanosoma brucei brucei parasite from the infected blood. we bought DEAE Cellulose from Biophoretics which i tried using but isnt working. the blood isn't retained in the resin
is there a special way to prepare this resin for elution?
what could i have done wrong?
Hello i am wondering if anyone has tried to cupture trypanosoma evansi in vitro?