Hallo! Does someone have experience culturing cells derived from infectious samples?
I would like to isolate the bone marrow mesenchymal stem cells from patients diagnosed with bone infection and culture them. The only problem is that the tissue would be infectious with bacteria inside. Some of the bacteria might be also multiresistant. If I culture them in the incubator, there might generate bacteria membrane, and even contaminate other cell wells. Is there any good idea how I can eliminate these bacteria and culture these stem cells?
1. You need to know which antibiotics, antimycotics, antifungals, etc., and at which maximal concentrations will not kill your cells. Suggest obtaining purified mesenchymal stem cells from commercial source. Obtain various sterile solutions of antibiotics, antimycotics, antifungals, etc. from various commercial sources. Plate the MSCs in 96-well plates at 10,000 cells per well in one-half plating medium. Then add 2nd half of medium containing a range of concentrations: 0%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2.5%, 5% (n=3) of various antibiotics, antimycotics, and antifungals. You should be able to run 4 different samples on a single 96-well plate. [Because the lab I was using at my institution was previously used by a microbiologist, we performed the same experiment. We discovered that a combination of Penicillin + Streptomycin + Fungizone was necessary in our cultures to prevent contamination].
2. Every time you obtain a sample of infectious material, you will need to screen the sample with all antibiotics, antimycotics, and antifungals, at maximal concentration, that will not kill your cells. This experiment is performed unless clinical lab has already determined susceptibility of infectious agent to particular drug(s) of choice. I would suggest using Ouchterlony plate assays. You Tube has a short video from BioRad on how to perform the assay. Place antibiotics, antimycotics, antifungals (maximal concentrations) in the four wells of agarose medium. Medium without any agents should be in center well. Grow a sample of your infectious sample on the agarose substrate. Culture at 37C for 3-4 days. Any well that has a clear zone around it that is absent of infectious agent, but with healthy cells is a keeper.
3. Mesenchymal stem cells from humans have particular cell surface cluster of differentiation markers, e.g., CD105, CD117, CD123, and CD166. You could either use one or all of these to obtain purified MSCs from bone marrow. Unless you have access to a cell sorter, I would suggest using Miltenyi magnetic bead system. The beads are significantly smaller than the size of MSCs and will be absorbed by the cells in a few days of culturing. The 'quick and dirty' method is to use all four CD markers attached to magnetic beads simultaneously. Be forewarned though, there are other cell types that have these markers individually on their cell surface. So you will probably be isolating more than just MSCs. A good source for CD marker identification is Cellular and Molecular Immunology by Abbas, Lichtman, and Pilai to determine which potential cells belong to which CD markers. Alternatively, the better method is to run the cells in sequence, first CD105, then CD117, then CD123, and finally CD166. While this method will give one higher purity, the yield will be significantly less. Perform all experiments (Miltenyi included) inside laminar flow hood (see below).
4. Lastly, and most important for you and others, if you share facilities.
A. Working with human cells, even if they are not infectious, you need to glove, gown, mask, face shield, booties, and hair net, inside your culture facility. You are protecting the cells from any flora you have and protecting you from the infectious agents the cells might have, as well as protecting you from the cells themselves. [In my first job, I was sharing space in a culture facility that routinely grew human chondrosacomas from biopsy specimens. The head of the lab refused to wear protective gear and would routinely pipet media by mouth. He came down with cancer ... chondrosarcoma. When genetically sequenced, his cancer was not from his cells that had mutated, but rather one of the chondrosarcomas he was culturing]. You should gown and ungown inside the culture facility. Place all used protective coverings in a biohazard bag and autoclave on dry cycle when finished. B. Only perform your work in a Class-II (or higher numbered) HEPA-filtered Laminar Flow Biosafety Cabinet. Preferably, hood has built-in UV light, which should be on at all times when someone is not working in the hood. Preferably, laminar flow should be running in hood 24/7. If not, laminar flow needs to be on 45 min before starting work. Work surface of hood should be wiped down with 70% Isopropyl alcohol before any work is performed in hood, after each set of experiments, changing medium between different cultures, etc. Wipe up any spills immediately with 70% alcohol dampened wipe (e.g., KimWipes, sterile gauze, etc.). When finished, hood should be liberally sprayed with Isopropyl alcohol and allowed to air dry. An alternative disinfectant to 70% isopropyl alcohol is Amphyll. Amphyll is a phenol-based disinfectant and can be used in same manner as the alcohol. The laminar flow hood should NOT be used as a storage area. Over time, UV radiation will cause failure of plastic materials, and alter characteristics of solutions, media, etc.
C. Dedicate the incubator(s) to your experiments ONLY, to prevent contaminating other cultures. Preferably the incubators are lined with either copper or zinc. Zinc flashing can be substituted if placed in a pan of water to keep incubator humidified. Sodium azide (5%) can be substituted for zinc flashing [be forewarned sodium azide will kill everything, including yourself]. If handling powdered form of azide, wear a respirator and nitrile gloves to handle it in a chemical fume hood when weighing out powdered form. If in aqueous form, inhalation is nonexistent, so only nitrile gloves need to be worn.
D. Any aspiration system for permanently removing liquids should be structured in such a manner that the liquids are siphoned off into a concentrated bleach solution. When bleach concentration is diluted to ~10% by volume, switch to new container, and autoclave removed container on wet cycle. Discard liquid after autoclaving.
E. Any pipets, plasticware, etc., used during culturing and discarded, should be discarded into a container of 10% aqueous bleach solution. When container full, remove items to biohazard bag and autoclave on dry cycle. Discard in trash when completed autoclaving. Spent bleach solution can be poured down drain.
Good luck with your experiments.
If you have any questions on any of the above or any other questions for that matter, please ask.
Dear Yi,
Can I ask you question: why you want MSCs from infected bone marrow? your purpose is isolating stem cells or experiment of infected stem cells,
Henry E Young Thank you Dr. Young with such detailed explanation and your reminders for safety issues! I have two questions here: 1. I am going to evaluate the influence of infection for BMSCs, that´s why I need to separate the cells directly from patients. I have concerns that if I use commercially sold stem cells, it would be hard to simulate the in vivo environment. Do you have any suggestions how to isolate the cells and culture them? Or do you have any models in mind that can simulate the inner inflammatory environment? 2. If I sort the cells out by magnetic beads, or FACS, can this eliminate the bacteria?
Yi Ren, I suggested to use commercially available MSCs from bone marrow to work out all your potential variables ahead of time with non-infected tissue. Better for you in long run. Once all variables with isolation, etc. are known and standardized, you can then move to the infected samples (you are not wasting precious material trying to discover what works best).
Once your variables are standardized, I would suggest applying to NDRI an NIH-sponsored group that provides any type of human tissue in any format. When we performed the study to determine whether our telomerase positive stem cells would decrease with increasing age, we obtained aged samples of human tissue for the experiment, e.g., newborn, puberty, 20s, 40s, 60s, 80s.
Yi Ren, I suggested to use commercially available MSCs from bone marrow to work out all your potential variables ahead of time with non-infected tissue. Better for you in long run. Once all variables with isolation, etc. are known and standardized, you can then move to the infected samples (you are not wasting precious material trying to discover what works best).
Once your variables are standardized, I would suggest applying to NDRI an NIH-sponsored group that provides any type of human tissue in any format. When we performed the study to determine whether our telomerase positive stem cells would decrease with increasing age, we obtained aged samples of human tissue for the experiment, e.g., newborn, puberty, 20s, 40s, 60s, 80s.
Yi Ren, we used a mixture of two enzymes (collagenase-1 and dispase) to isolate cells from solid tissues. The exact protocol for isolation, plating, and culturing was already given in an earlier question. Just search my name under questions.
It is doubtful that you will lose infectious agents during cell sorting, but I could be wrong. To test that parameter, again use commercially available MSCs and mix with a known quantity of an infectious agent (can obtain from ATCC). Mix the two, then run over a sorting column or cell sort. Take resulting fraction and plate on microbiological plates (Thermo-Fisher Scientific at 25C and 44C. And see if any bacteria grow out from sample. And you have your answer.
Neither FACS nor Miltenyi columns will give 100% purity of cell type. But either should give you around 90% purity.
You will probably need to use an antibiotic, antimycotic, antifungal mixture in all your solutions, e.g., culture medium, buffers, enzymatic digests, freezing medium, etc., to prevent continued contamination of your cultures.
Most make the mistake of only including the ‘anti-x’ in medium and then become frustrated with continued contamination. If present in all solutions that the cells/contamination see, it prevents them from taking over the cultures.
Dear Ren,
In addition to outstanding recommendations from Henry, I would recommend changing media frequently, like everyday if possible until you see very clear and clean growth of cells with no bacteria. It will help to dilute the bacteria and wash them away from cells. If encountered contamination and it's a precious sample, try to treat with 2x concentration of antibiotics and anitmycotic. Cells can recover from bacterial induced changes in long term if you get rid of contaminants by doing so.
Hope it helps!
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