Hello Ilene.

Are you recovering stem cells from animals or human subjects?

It would be more controllable to isolate without contamination from small lab animals. Human stem cells are notorious for contamination due to many proper clinical procedures and the involvement of other people ( not the experimenter). However, general sterile techniques should be followed by yourself as follows;

1) Make your own new fresh solutions (including buffers, reagents and other culture supplements. Careless preparation of certain supplements stored at deep freezer or fridge would jeopardized one's culture even if precocious cares are taken by adding the frozen and thawed supplements to the well-managed sterile culture.

2) Any solutions for washing, digestion, and medium should be maintained properly without unexpected contamination opportunity.

3) Centrifuge frozen-thawed reagents, or digestion solutions (T-EDTA sol, collagenase, DNase and etc.) at 10,000xg for 10 min at 4C to precipitates microbes and other particles at the bottom of the Eppen-tubes alliquoted.

4) For the time being, make fresh everything weekly and through them away when not used.

5) Gas lamp ( alcohol lamp) and 70% EtOH spray are necessary and wash your hands with soup extensively and then wear gloves before do anything.

6) Sterilize the CO2 incubator by any fuming methods or 70% EtOH spray and several times. Contaminated incubator would allow microbes get in the culture dishes or flasks by the fanning forces through the lid or tops.

7) Test microbial contamination whether the transferred tissues or samples from collectors are already contaminated with sterlile buffer washed solutions collected, centrifuged and microbial culture of the pellet-suspension spread on agar plates. This is most likely the source of the contaminant if you are not the cell or tissue collector.

Study any good cell culture books.

Good luck.

I am using human umbilical cords.

Thank you so much for your help! I think I will try the centrifuge first, because I am thinking its from heat inactivated FBS in media* prepared by former lab member.

I will also start using gas lamp.

Hi Ilene,

I agree with all the suggestions given by Prof. Sang Ho Lee except point 5. Please don't use gas lamp inside the bio-safety cabinet.

Best wishes

Hello

I agree with all researchers ; Do you transport the umbilical cord sample by transport medium ( DMEM with 2 % Pen/Strep )? also should be add antibiotic to your culture to clean them up. but If you are already using Pen/Strep you should add a different antibiotic but some antibiotics interfere with cell function and with a bacterial infection you cells will generally look unhealthy, the media may change color or turbidity, and through the microscope you will see moving black dots or rods in the media and the cells , for that should be carefuled when you use it

Good Luck

I agree with all suggestions given by Prof. Sang Ho Lee

Dear Ilene,

I agree with Prof Lee, but there is an additional factor that you have to take into account.

Is the umbilical cord from Caesarean section or normal delivery?

If from Caesarean section it is pretty clean, but if from normal delivery, the cord has passed a route full of contaminants, so you need to decontaminate the cord before processing.

Immerse in Povidone iodine -PBS solution several times, then in 70% ethanol 3 times, and finally in PBS. Then discard the very end of the tissue before processing further.

Good luck

I do not collect the cord myself so I have no way of finding out from delivery or C-section. I do wash with betadine and 70%EtOH before processing.

And I have found out its not bacterial contamination. It looked so under microscope, but it was tissue debris since number does not increase after 7 days. Thank you all for suggestions!

I was going to tell you that, after enzimatic digestion you are going to have debris! You could also try explant culture, its cheaper and in my opinion it has similar or even better results. Eventually you will observe a lot of small dots in some of your cultures, that usually happens and its not related to contamination, I believe they are exosomes

Thank you Dr. Raffo! I will wash those cells once enough of them attach to surface.

Use gentamycin. In our lab people working with human tissue explants always eliminate bacteria via gentamycin.

As we all know, anything you get from a hospital is probably contaminated! All good answers - a pre-sanitization step of the external tissue with povidone or equivalent is a must, and wash off the povidone with PBS containing 2x P/S amphotericin (a fungiside, but worth including). Alcohol has minimal germicidal activity unless you allow it to dry in situ and remember that germicides such as povidone have minimum effective contact time be sure wait for that minute before cutting! I also recommend slicing and discarding the ends of the specimen with a sterile scalpel before processing the tissue to reduce the chances of bacteria contaminating the ends that do not see disinfectant. Finally ... are you sure the cultures are contaminated by bacteria?

Thank you Dr. Carl! No, I do not think its really contamination after culturing those cells for over week. Those particles remain floating and did not increase in number. I washed them off as most cells now attached to plate and spread. My only concern now is black particle attached to cells that will not be off with washing. It could be exosome, but I'm not 100% sure of it. Its present in almost all cells I culture. Most likely due to using old FBS or other components. Unfortunately, we cannot afford to purchase some of the materials, so it will definitely affect our work.

It can be sound strange but these black particles can be out of the plates. Sometimes in incubator metal surface can generate these dust like particles. If possible could you please clean the bottom part of the plate with a napkin and alcohol from the out surface?

Thank you for the suggestion! I will try cleaning the plates before changing media from now on.

Hi, all the above suggestions are valuable, and knowing that these small particles do not move, I assume that they should be apoptotic bodies for cells that have died or microvesicles. Exosomes are nano-sized and need electron microscope to be seen.

If you are saying that it is due to old FBS and other components, then try to centrifuge before using to pellet these particles.

All the best.