JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

Essentials of
Neuroscience

You do not have subscription access to videos in this collection. Learn more about access.

Essentials of Developmental Biology

You have subscription access to videos in this collection through your user account.

 JoVE Biology

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

1, 1, 1, 1

1Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco

Article
    Downloads Comments Metrics

    You must be subscribed to JoVE to access this content.

    This article is a part of   JoVE Biology. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.

    Recommend JoVE to Your Librarian

    Current Access Through Your IP Address

    You do not have access to any JoVE content through your current IP address.

    IP: 54.80.69.152, User IP: 54.80.69.152, User IP Hex: 911230360

    Current Access Through Your Registered Email Address

    You aren't signed into JoVE. If your institution subscribes to JoVE, please or create an account with your institutional email address to access this content.

     

    Summary

    This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

    Date Published: 4/02/2008, Issue 14; doi: 10.3791/686

    Cite this Article

    Boddington, S., Henning, T. D., Sutton, E. J., Daldrup-Link, H. E. Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging. J. Vis. Exp. (14), e686, doi:10.3791/686 (2008).

    Abstract

    Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

    Protocol

    Labeling of mesenchymal stem cells with the fluorescent dye DiD

    1. To begin the procedure for labeling mesenchymal stem cells, trypsinize and count the cells to get a suspension with a defined number of cells.
    2. Take the cells out of the incubator and aspirate out old media from the flasks containing the cells to be labeled
    3. Wash the cells with 10 ml of Mg/Ca-free PBS. Aspirate out the PBS. The Mg and Ca would inhibit the Trypsin, therefore we use PBS without these.
    4. Add pre-warmed 0.05% Trypsin, for a T75 flask we use 5ml. Ensure that the entire surface of the flask is covered.
    5. Incubate at 37°C in the incubator for about 5 minutes.
    6. Confirm the detachment under the microscope. If the cells still adhere to the flask, tap it a few times and wait a little longer until the trypsinization is successful.
    7. Now, it is necessary to neutralize the Trypsin by adding media containing 10% FCS. We use an equal amount of media as there is Trypsin.
    8. Pipette up and down a few times to ensure that all the cells are re-suspended in the media.
    9. Transfer the cell solution to a sterile 15ml capped polypropylene tube.
    10. Centrifuge at 400 rcf for 5 minutes.
    11. Aspirate out the supernatant ensuring not to disturb the cell pellet.
    12. Resuspend the cells in DMEM and proceed with the cell count.
    13. Suspend the cells to be labeled at a density of 1x10^6 per ml in serum-free culture medium (DMEM).
    14. This was all preparation of the cell suspension. Now, it is ready to start the labeling.
    15. First, add 5 µL of DiD contrast agent per ml of cell suspension.
    16. Then, mix the solution by gently pipetting.
    17. Incubate the cells with the labeling solution at 37°C for 20 minutes in a 6-well low-attachment dish.
    18. Once the simple incubation is complete, it is necessary to transfer the cell solution to a 15 ml capped polypropylene tube.
    19. Centrifuge it down at 400 rcf for 5 minutes.
    20. Aspirate out the labeling medium, ensuring not to disturb cell pellet
    21. Wash the cells with PBS. Pipette the cells up and down, making sure to break up the cell pellet.
    22. Repeat the latter two steps two more times, so there is a total of 3 washing steps.
    23. Count the cells and perform a Trypan blue test to determine cell viability These are standard cell culture protocols that we are not going to explain here.
    24. Your cells are now ready to be imaged in optical imager.

    Labeling of human embryonic stem cells with the fluorescent dye ICG

    1. To begin the procedure for labeling human embryonic stem cells, prepare the contrast agent Indocyanine Green. Mix it with the transfection agent Protamine.
    2. Measure out 1mg of the Indocyanine Green powder. Dissolve the ICG powder in 100 µL of Dimethyl Sulfoxide (DMSO).
    3. Add 400 µL of Dulbecco's Modified Eagles Medium (DMEM + 10% Fetal Calf Serum) media to the mixture and shake it well. This results in a final concentration of 2mg/ml of Indocyanine Green.
    4. Add the transfection agent Protamine. Protamine acts as a shuttle for the contrast agent, so that it gets into the cell more efficiently.
    5. Mix 5 µL Protamine Sulfate, which is supplied at a concentration of 10mg/ml, with 300 µL ICG and 300 µL serum free Dulbecco's Modified Eagles Medium.
    6. Gently shake the new transfection solution for 5 minutes to allow complex to form.
    7. The labeling solution is ready.
    8. Aspirate out the old media from hESC 10mm Petri dish.
    9. Add 5ml of pre-warmed serum-free DMEM.
    10. Add the previously prepared Protamine/ICG solution to the cells. Start the 1 hour incubation by placing the dish in an incubator at 37°C.
    11. After the incubation is complete, remove the dish from the incubator and aspirate out the labeling solution.
    12. Wash the cells by rinsing the dish with 5 ml PBS.
    13. Aspirate out the PBS and replace with 5ml of 0.25% Trypsin. Incubate the dish at 37°C for 5 minutes to allow trypsinization to occur. It helps to shake the dish a little every once in a while.
    14. Gently pipette up and down, to break up the remaining colonies.
    15. Neutralize the Trypsin by adding an equal amount of KSR to the dish.
    16. Transfer the cell solution to a 15ml tube and centrifuge the solution at 400 rcf for 5 minutes.
    17. Resuspend the cells in full media.
    18. If there still are clumps at this point, trypsinize and centrifuge again.
    19. Once a clump-free cell solution is achieved, aspirate out the old media and resuspend the pellet in 10ml of pre-warmed full ESC media.
    20. At this point, it is necessary to separate the mouse feeder cells from the hESCs. This is done to ensure that, later, only imaging the stem cells occurs.
    21. For this, transfer the cell solution to a gelatin coated 10 cm dish.
    22. Put the dish into the 37°C incubator and let it sit for 45 minutes. During this time, make sure not to disturb the dish.  Now, the feeders will adhere to the dish and the stem cells will not.
    23. Transfer the solution out of the Petri dish. and now we have a labeled single cells solution of human embryonic stem cells.
    24. The cells can now be counted and a Trypan blue test can be performed on them.
    25. The cells are ready to be imaged!

     

    Subscription Required. Please recommend JoVE to your librarian.

    Discussion

    OI is a relatively new imaging technique, based on the detection of fluorescence. OI is as sensitive as radiotracer-based imaging techniques, but not associated with any irradiation exposure. OI provides an effective means of tracking cells non-invasively and repetitively, thereby providing insight into cell migration to the target site. One major limitation of the technique is the limited tissue penetration of fluorescent probes in vivo. Thus, a tracer accumulation in deep tissues, more than about 5-10 cm from the skin surface, may not be detected. Current clinical applications are limited to superficial techniques such as endoscopic, cardiovascular and retinae imaging (Funovics et al. 2003) but this domain of research will continue to expand through the recognition of the vast possibilities offered by in vivo cell tracking.

    Subscription Required. Please recommend JoVE to your librarian.

    Disclosures

    Acknowledgements

    This project was supported by a Leon J. Thal SEED grant from the California Institute for Regenerative Medicine. Tobias Henning was funded by a Research stipend from the German Research Association (DFG, HE 4578/1-2). We want to gratefully acknowledge Juanito Meneses for his advice on the culture of human embryonic stem cells.

    Materials

    Name Type Company Catalog Number Comments
    Indocyanine Green (IR-125) Reagent Fisher Scientific AC41254-1000
    DMSO Reagent Sigma-Aldrich D-2650
    D-MEM High Glucose Reagent Sigma-Aldrich D5648
    Gelatin Reagent Sigma-Aldrich G1890 Dilute to final concentration of 0.1% in PBS
    PBS, Mg and Ca free Reagent GIBCO, by Life Technologies 14190-144
    Trypsin-EDTA 0.05% Reagent Invitrogen 25300-120
    Trypsin-EDTA 0.25% Reagent Invitrogen 25200-114
    FCS, characterized Reagent Hyclone SH30071.03
    Cell Strainer (40 μm Nylon) Reagent BD Biosciences 352340
    DiD Reagent Molecular Probes, Life Technologies V-22887
    Knockout DMEM Reagent GIBCO, by Life Technologies 10829018
    Knockout Serum Replacer Reagent GIBCO, by Life Technologies 10828028
    b-Mercapt–thanol Reagent Sigma-Aldrich 7522
    Non-essential Amino Acids Reagent GIBCO, by Life Technologies 11140050
    FGF-2 Reagent R&D Systems 233-FB-025
    Glutamine 200mM Reagent GIBCO, by Life Technologies 25030081
    Penicillin/Streptomycin Reagent GIBCO, by Life Technologies 15140-122
    Protamine Sulfate Reagent American Pharmaceutical Partners

    References

    1. Daldrup-Link, H. E., Rudelius, M., Metz, S., Piontek, G., Settles, M., Pichler, B., Heinzmann, U., Weinmann, H. J., Schlegel, J., Link, T. M., Rummeny, E. J., Oostendorp, R. A. J. Stem cell tracking with Gadophrin-2 -- a bifunctional contrast agent for MR imaging, optical imaging and fluorescence microscopy. Eur J Nucl Med Mol Imaging. 31, 1312-1321 (2004).
    2. Simon, G. H., Daldrup-Link, H. E., Kau, J., Metz, S., Schlegel, J., Piontek, G., Saborowski, O., Demos, S., Duyster, J., Pichler, B. J. Optical imaging of experimental arthritis using allogeneic leukocytes labeled with a near-infrared fluorescent probe. Eur J Nucl Med & Mol Imaging. 33, 998-1006 (2006).
    3. Sutton, E., Henning, T., Pichler, B., Bremer, C., Daldrup-Link, H. E. Cell Tracking with Optical imaging. Eur Radiol. 2, 350-357 (2003).
    4. Funovics, M. A., Alencar, H., Su, H. S., Khazaie, K., Weissleder, R., Mahmood, U. Miniaturized multichannel near infrared endoscope for mouse imaging. Mol Imaging. 2, 350-357 (2003).

    Comments

    5 Comments

    Hi,Congratulations on the excellent video article. Please allow for two questions: 1. how many generations until the dye is diluted below the point of detection with the respective procedures ?  ². do you see directed movement in lesion models (stroke etc.)?Thanks,Peter
    Reply

    Posted by: AnonymousApril 4, 2008, 2:08 PM

    I am very happy after ateending your talk...but I need your help. I will plan to start to culture mesenchymal cell in he lab.can you send me detail protocol in which you mention each and every step and also standerd cell culture step.
    Reply

    Posted by: AnonymousMay 4, 2008, 2:20 PM

    Peter, Sushma, the author indicated to us that they prefer communication through email.  Please feel free to send email directly to daldrup@radiology.ucsf.edu.
    Reply

    Posted by: AnonymousMay 5, 2008, 8:17 PM

    Can we use the same protocol for DiO fluorescent dye because we need green fluorescence
    Reply

    Posted by: AnonymousJuly 7, 2009, 11:08 AM

    Hello Dr. Daldrup-Link team,
    It was great seeing this video. Great job guys.
    I am wondering if you can help me answering some of my questions for the DiD labelling.

    For how long I can see the signal in vivo (mice) after IP injection? Have you tried it for weeks?
    Can I still use the DiD for IHC imaging of the Parafin embedded sections?

    Thanks,
    Osama
    Reply

    Posted by: Osama M.June 26, 2012, 3:20 PM

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter