I am growing bladder cancer cells in 3D models in vitro and I wish to visualize them using some form of real-time imaging which will still allow me to grow them and use them for protein/RNA extraction. Any suggestions?
ِDear Naomi
Let me know,have you any experience about the Holomonitor M4?
Sincerely
Ahad
Depends on how long you want to image them for. If its for a longer periods you need a microscope equipped with a 37C and 5%CO2 chamber, so that the conditions are similar to the incubator. If your microscope is sterile then u can handle your cells normally afterwards.
We have a Nikon Eclipse Ti that we have used to monitor connective tissue cells proliferating in 3D collagen scaffolds. The microscope produces time-lapse videos and has a heated/gassed stage for multiwell plates so the cultures are just as happy on the stage as they are in the incubator.
Hi Naomi--take a look at HyStem-C from ESI Bio. It's transparent, allows you to image the cells in a lifelike matrix, and then recover the protein or RNA for subsequent analysis. We've used it quite a lot for making 3D tumor models.
Any medium quality confocal microscope with an appropriate stage to load microplates or multichamber slides and phase capability. You may also wish to consider Optical Computer Tomography if you only need an indication of cellular density and location within a 3D culture but not be too concerned about detail.
Hi Naomi - What is the format of your cultures? Are they overlaid or embedded in basement membrane? And are they in some kind of glass-bottom dish (e.g. MatTek or glass-bottom multiwell)? As long as you are simply imaging them with transmitted light using a standard contrast enhancement technique (phase contrast, DIC, darkfield, etc) and you are not staining with any fluorescent labels that require terminal fixation, then you can certainly just image them with your inverted microscope, then put them back in the incubator and let them keep growing until you are ready for terminal protein/RNA quantification. As at least one other here commented also, if it is your goal to monitor growth over extended durations (hours, days) then of course you need a time lapse microscope with appropriate enclosure for controlled environment (37C, 5% CO2). There are also more advanced techniques for longitudinal 3D imaging that you could look into: digital holographic microscopy, OCT etc. If you are interested we have a few papers that deal with longitudinal imaging of various in vitro 3D tumor models, both using snapshots at discreet timepoints and timelapse monitoring.
Hi Naomi,
I have worked with explant tissue culture system, and I used to regularly image the tissue and cells with a widefield (inverted) microscope. I was looking at cell-matrix changes following tissue disruptions, and I aborted my experiments at 36 hours,. As a rule, the widefield microscope had an incubator around it and the environment of the culture system was maintained at 37 degree centigrade with 5% carbon dioxide and, 95% air. The wide field microscope, was fitted with a computer that had Volocity software within it, the software allowed me to monitor the tissue and cellular changes like a time lapse microscope (I used to spend hours under the microscope recording video of cellular movements following matrix damage). Just to let you know, the tissue was kept in a petri dish with a glass bottom, plastic dish tend to create artifacts hence its always best to use petri disc with glass bottom ( Mat tek is the best, which we use to order all the way from United States).
Please note, when I didn't monitor changes in the tissue explant, I used to keep it in an incubator ( maintained at 37 degree, and 5% CO2).
After aborting the experiment, I used to do immunofluorescence analysis to look at specific protein of interest, but I can assure you that you can use the tissue/cells in 3-d culture to extract protein as well.
I have a paper describing the culture system and live cell imaging technique used in that study, which has been appreciated as a special poster for an International conference.
Hope this helps and all the best for your study.
Polly
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