Hello Luisa, we saw that too. Often, it is because the culture was frozen at a too high (or too low) density. Check cell density before freezing, it may vary from one strain to the other though, for example the 29-13 cells need to be frozen at a higher density (10exp7) than other 427 derivatives (5.10exp6). Glycerol being viscous, we filter the freezing solution. Finally, when we thaw the cells, we do a couple of transitions (one wash at 4°C, a second wash at RT) before putting them in culture. Check also the density once back in the flask as cells can be lost in the centrifugation steps. Good luck! Philippe

Dear Loiusa,

I use a protocol for freezing trypanosomatids that works perfectly. You should freeze ~10(7) parasites in 90% FBS + 10% DMSO. I use to prepare the mix on ice and incubate at -80 overnight in NALGENE Mr Frosty Cryo Freezeng container (containing IPA). Next day you can storage your vials in the nitrogen tank.

Best wishes!

Hi Luisa

When freezing cells, I make stock concentration of 14% glycerol in SDM79 or cunningham medium (whatever you are use), filter sterile and keep at 4C. I usually freeze cells from culture of 5000,000 cell/ml density (procyclic) or 100,000 cell/ml (bloodstream). After harvesting the cells, I re-suspend them in final conc. of 7% glycerol/medium, stock the vials in slow -80 cooler (1C/min). I revived viable cells after 3 months by using this way, but it is always safer to keep cells in liquid nitrogen.

Good luck

I agree with Mohamed's comment, we rarely managed to revive vials that had been kept for more than 18 months at -80°C. In contrast we recently thawed the snl-2 trypanosome cell line that had been stored in liquid nitrogen since 2001 and cells were perfectly fine!

Hi

Try this few steps, it work nicely with me

1. Inject a mice with your isolate, monitor it daily for approximately 4 days (parasitamia will reach about 10 million per milliter)

2. Harvest the parasite via cardic (about 1.5ml)

3. Mix 0.4ml with 0.1ml 60% cool glycerol

4. Keep the mixture in -80 overnight

5. Transfer it to LN.

I kept my parasite for almost 2 year in LN & still survive.

Try to make at least 2 vials for each isolate

Best luck

Sh

I forgot to say that the above comment was for procyclic stage trypanosomes

Hello, I also used the same method like Elshafie Hassan. The viability of parasites was very active and nice for more than 12 months, even some are death but not more than 10% death. However there is some point different; I used coagulation (Sodium citrate) when I collected blood from heart. Then mixed with 30% glycerol in PBS (pH7.4) (Steriled is recommended).

Have a nice results!

Good luck

Ketsarin Kamyingkird

I did not mention this in my initial question, but our problems are with bloodstream forms from culture. Based on your comments, we will filter the glycerol freezing solution and we will buy a Mr freezing unit. We supposedly pay attention to the cell density before freezing, but I will remind my group of it. We also use liquid nitrogen for long term storage.

Iam sorry iam not understand

Hi Luisa,

I routinely use a simple protocol. Add 900ul BSF cell culture (about 1,000,000 cells) to 100ul glycerol and mix gently using a 1ml tip, then place at -80c for 24h, then store long term in liq N2. To thaw, hold at 37c for a few minutes and transfer whole vial to 10ml HMI9/10%FBS at 37c - there's no need to spin out the glycerol or to use an initial low temp incubation. I've not had problems with glycerol, but then we make lot of cell lines, so we use it up fairly quickly. Typically, problems thawing are a consequence of poor mixing prior to freezing - either insufficient or too vigorous. Hope this helps.

Should've said, I place the cryo mix straight into the -80c freezer, so they freeze fairly quickly but not instantaneously. I've also frozen the same mix on dry ice, which is probably a lot faster, and had no problems with viability. Basically, I don't worry too much about controlling the speed of freezing.

Trypanosome metabolism is completely inhibited by anaerobiosis in combination with glycerol, exactly the conditions that are prevalent in the mixtures proposed for the freezing of the bloodstream forms of the African trypanosomes. Therefore I recommend to use DMSO rather than glycerol as cryoprotecting agent. The reason for this is that in the case of bloodstream forms in the absence of oxygen glycerol, above a concentration of a few millimolar, completely blocks glycolysis, which is the only ATP generating pathway for these cells (see Fairlamb, Opperdoes and Borst, Nature 1977, 265:270-271, . When you thaw the cells after storage and the temperature is raised above zero degrees centigrade the cells want to speed up their metabolism but will starve from a lack of ATP. This explains the highly variable results of viability of the cells when one does not take care of the temperature before freezing and after thawing.