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

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

1, 1

1Department of Chemical Engineering and Materials Science, City of Hope Cancer Center

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: 54.166.105.24, User IP: 54.166.105.24, User IP Hex: 916875544

    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

    Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

    Date Published: 3/22/2010, Issue 37; doi: 10.3791/1852

    Cite this Article

    Zhang, L., Chan, C. Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs. J. Vis. Exp. (37), e1852, doi:10.3791/1852 (2010).

    Abstract

    MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

    Protocol

    1. Isolation of Rat MSCs

    Mesenchymal stem cells were isolated from 6-8 week old Sprague-Dawley female rat as previously described 1, 2. Isolated MSCs can adhere to plastic surface and easily expand during in vitro culture.

    1. The animal was put into an anaesthesia chamber and anaesthetized for around five minutes. During the anaesthesia, observe the rate of blinking, breathing and motor activity. Remove the animal from the chamber immediately after it stops motor activity and the blinking rate became infrequent.
    2. Lay the animal down on an operation station and kill the animal by cervical dislocation. Cut off the femurs and tibias from the back limbs, remove the skin and muscles.
    3. Put the dissected femurs and tibias in 70% isopropanol for a few seconds and transfer to 1X D-PBS.
    4. In a biosafety cabinet, the femurs and tibias were transferred to a 10cm dish containing DMEM. Each bone was then held with tweezers and the two ends were cut open with a scissor. Attach a 22G needle to a 3ml syringe and fill it with DMEM, then flush the marrow into a 50ml tube by inserting the needle to one open end of the bone. Repeat 2~3 times for each bone. When all the marrows were obtained, resuspend the cells and pass the cell suspension through a 70μm cell strainer to remove the bone debris and blood aggregates.
    5. Spin down cells at 200g, 4°C for 5 minutes and remove the supernatant by aspiration. Resuspend cells in 25ml MSC medium (DMEM containing 10% FBS and 1% Pen-Strep). 10ml cell suspension was seeded into each 10cm culture dish for a total of two dishes. Keep the culture dishes in a 37°C and 5% CO2 incubator for 1~2 weeks. Medium was changed every 2~3 days.

    2. Enrichment of Rat MSCs

    A number of surface proteins have been used to enrich MSCs, including CD54, CD90, CD73, CD105 and CD271 3-5. In our study, we used CD54 and CD90 as markers to enrich MSCs by magnetic cell sorting.

    1. When cells reached around 80% confluency, aspirate the medium and add 4~5ml trypsin-EDTA to each dish. Put the dishes back to the incubator and incubate for around 5 minutes to allow cell detachment. Once cells were detached, add equal amount of culture medium to inactivate trypsin Collect cell suspension into a 15ml tube and spin cells down at 200g, 4°C for 5 minutes.
    2. The next steps describe how to enrich MSCs by two surface markers CD54 and CD90 according to the manual for cell separation using BD IMagnet. Cell pellet was resuspended in cell staining buffer (3% heat inactivated FBS in 1X D-PBS) at 20 million cells/ml. Biotinylated CD54 antibody (0.25μg per million cells) and biotinylated CD90 antibody (0.15μg per million cells) was added and mixed gently with the cell suspension. After incubation on ice for 15 minutes, labeled cells were washed with an excess volume of 1X BD IMag buffer. The labeled cells were spun down at 200g, 4°C for 5 minutes.
    3. Vortex the BD IMag streptavidin particles thoroughly, add 40μl particles for every 10 million cells. Mix the particle with the labeled cells thoroughly and incubate the cells at 6~12°C for 30 minutes. This allows the streptavidin particles to bind to the biotinylated anti-CD54 and anti-CD90, which is bound to the surface proteins CD54 and CD90 respectively.
    4. During the incubation time, label a round-bottom test tube to collect the positive fraction. After incubation, bring the labeling volume to 20 million cells/ml with 1X BD IMag buffer and transfer the labeled cells to the positive-fraction collection tube. Place the positive-fraction tube onto the BD IMagnet and let it stay for 6 minutes, then remove the supernatant with a glass Pasteur pipette with the positive-fraction tube still on the BD IMagnet.
    5. Remove the positive-fraction tube from the BD IMagnet and place it on ice. Add 1ml ice cold 1X BD IMag buffer and resuspend cells by gentle mixing. Place the tube back onto the BD IMagnet and let it stay for 2~4 minutes. Remove the supernatant with a new glass Pasteur pipette. Repeat the washing as described in 2.5 one more time.
    6. Remove the tube from the BD IMagnet and resuspend cells in culture medium, seed one 75cm2 flask for maintaining the cells and a 10cm dish for flow cytometry.

    3. Verification of Surface Marker Expression by Flow Cytometry

    Flow cytometry analysis was performed to verify the cells we obtained express CD54 and CD90. The HSC marker CD45 was used to confirm that the MSCs were depleted of HSCs.

    1. When cells become ~80% confluent, trypsinize cells with trypsin-EDTA and collect cells into a 15ml tube. Centrifuge at 200g, 4°C for 5 minutes to collect cells.
    2. Aspirate the supernatant and wash cells with 1X D-PBS once. Resuspend cells in cell staining buffer (1X D-PBS containing 2% FBS and 0.05% sodium azide) to a final concentration of 5~10 million cells/ml and keep cells on ice. Aliquot 100μl cell suspension into the six labeled tubes (1. cells only; 2. isotype control IgG2a; 3. Isotype control IgG1; 4. CD45; 5. CD54; 6. CD90).
    3. Add isotype controls and primary antibodies at appropriate concentrations (IgG2a and IgG1: 20μl per million cells; CD45: 0.5μg per million cells; CD54: 0.25μg per million cells; CD90: 0.15μg per million cells) and incubate at 4°C for 30 minutes.
    4. Wash cells with 1X D-PBS twice and then resuspend cells in 100μl cell staining buffer. Add SA-PE (streptavidin- phycoerythrin, use 0.15μg per million cells) and incubate at 4°C for 30 minutes in dark.
    5. Wash labeled cells with 1X D-PBS twice. Resuspend cells in 400μl cell staining buffer and transfer to a falcon tube for flow cytometry analysis.

    4. Separation of Single-colony Derived MSCs

    MSCs is a heterogeneous population composed of different subpopulations with different cell shape, growth rate as well as differentiation ability 6. However, all the subpopulations express the known MSC markers and therefore it is not feasible to use markers to separate out these subpopulations. Therefore, we applied cloning cylinders to separate out the different subpopulations, which are colonies formed by single cells.

    1. Plate cells at about 50~100 cells per 10cm dish. Incubate in a 37°C and 5% CO2 incubator for 1~2 weeks. During this period, examine well isolated colonies with an inverted microscope. Once the colonies have reached big enough size (better more than 100 cells in each colony), mark the colonies with a sharpie at the bottom of the dish. When pick the colony to be marked, make sure there are no surrounding colonies near the picked one.
    2. Aspirate medium and wash the dish once with 1X D-PBS. Pick up a sterile cloning cylinder with a sterile forceps and gently place it around the marked colony. Repeat this until all the marked colonies has a cloning cylinder placed over. The picked colonies should be far away from each other such that every cloning cylinder only contains one colony.
    3. Add 100μl trypsin-EDTA to each cloning cylinder and put the dish back to the incubator for ~5 minutes. After 5 minutes, check the cells under the microscope to see whether they are rounding up. When the cells have lifted up, add equal amount of culture medium to inactivate trypsin. Mix the cell suspension with a 200μl micropipettor and transfer the cell suspension to a 60mm dish containing 3ml prewarmed culture medium. Label the dishes properly and put them back into the incubator. Tracking morphological change over the next few days.

    5. Representative Results

    According to the protocol described in the part for rat MSCs isolation, plastic adherent MSCs should be visible the next day after plating. As cells continue to proliferate, the confluent cells should look like the cells shown in Figure 1A. When cells reach ~80% confluency (Figure 1B), subculturing can be carried out. During subculture, trypsin-EDTA was used to detach cells and lifted cells are small and round as shown in Figure 1C.

    Figure 1
    Figure 1. Phase contrast images of rat MSCs. (A) Confluent MSCs. The majority of cells are spindle-like or star-like. (B) MSCs around 80% confluency. (C) Lifted cells after trypsinization are small and round.

    Once MSCs are enriched by magnetic cell sorting, flow cytometry analysis is performed to verify the surface marker expressions. If the enrichment is good, the cells should show positive staining against MSC markers CD54 and CD90 but negative against the HSC marker CD45 (Figure 2). Isotype controls IgG2a and IgG1 are used as negative controls.

    Figure 2
    Figure 2. Flow cytometry analysis of MSCs for surface markers. MSCs were labeled with antibodies against IgG2a (isotype control 1), IgG1 (isotype control 2), CD45, CD54 and CD90.  MSCs expressed CD54 and CD90 but not CD45.

    When cells are seeded at proper clonal density, colonies should rise from single cells. Figure 3A represents a colony formed by a single cell. Cloning cylinders can then be used to separate the colonies and cells derived from the colonies can be cultured separately. Figure 3B and 3C represents cells derived from two individual colonies. The cells derived from colony 1 are spindle like (Figure 3B) whereas the cells derived from colony 2 are round (Figure 3C).

    Figure 3
    Figure 3. Colony formation by MSCs and single-colony derived cells. MSCs cultured at clonal density form individual colonies. These colonies can be separated by cloning cylinders and cells from different colonies can be cultured separately. (A) A representative colony formed by MSCs when plated at clonal density. (B) Spindle-like cells derived from one colony. (C) Round cells derived from another colony.

    Subscription Required. Please recommend JoVE to your librarian.

    Discussion

    This protocol describes how to isolate and enrich MSCs. A method to separate the single-colony derived cells is also incorporated. There are several steps that are important for a successful isolation, enrichment and colony separation. While doing cell isolation from rat, it is recommended to filter through a cell strainer or a sterile nylon mesh of similar size to get rid of the blood clots and bone debris. After plating the cells overnight, many dead cells will be floating in the medium and the dead cells are removed by replacing with fresh medium which should help the growth of the attached cells.

    The magnetic cell sorting in this protocol describes how to perform a positive selection, and similar procedures can be used to perform a negative selection. The amount of antibodies to be added may differ and optimization is required to achieve better sorting. This is also true for labeling the cells for flow cytometry analysis. If not running flow cytometry analysis for the labeled samples immediately, samples can be fixed with 2% formaldehyde and run later. However, long-term storage is not recommended since this tends to increase the auto-fluorescence and sacrifice sample quality.

    The key part for the colony separation is seeding at the right cell density (which should be optimized experimentally) and locating single clones that are not surrounded by other clones. If there are other clones nearby, the cloning cylinder may encompass the nearby clones and the cells obtained will no longer be from one clone When placing the cloning cylinder over the clone, also be careful not to slide it over the dish surface as this will cause the silicon grease at the bottom of the cloning cylinder to cover the cells and prevent the trypsin from reaching the cells to detach them.

    Subscription Required. Please recommend JoVE to your librarian.

    Disclosures

    Acknowledgements

    The work was supported in part by National Institute of Health (R01GM079688, R21RR024439, and R21GM075838), National Science Foundation (CBET 0941055), and the MSU Foundation.

    Materials

    Name Company Catalog Number Comments
    1X D-PBS Invitrogen 14040-133
    DMEM Invitrogen 11885-084
    Cell strainer BD Biosciences 352350
    FBS Invitrogen 26140-079
    Pen-Strep Invitrogen 15140
    Trypsin-EDTA Invitrogen 25200-056
    BD Imagnet BD Biosciences 552311
    Biotin mouse IgG2a BD Biosciences 555572
    Biotin mouse IgG1 BD Biosciences 555747
    Biotin anti-rat CD54 Cedarlane Labs CL054B
    Biotin anti-rat CD90 Cedarlane Labs CL005B
    Biotin anti-rat CD45 Cedarlane Labs CL001B
    BD Imag buffer BD Biosciences 552362
    Round-bottom tube BD Biosciences 352063
    Streptavidin particles BD Biosciences 557812
    SA-PE R&D Systems F0040
    Cloning cylinder Sigma-Aldrich C2059

    References

    1. de Hemptinne, I., Vermeiren, C., Maloteaux, J. M., Hermans, E. Induction of glial glutamate transporters in adult mesenchymal stem cells. J Neurochem. 91, 155-166 (2004).
    2. Porter, R. M., Huckle, W. R., Goldstein, A. S. Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells. J Cell Biochem. 90, 13-22 (2003).
    3. Chamberlain, G., Fox, J., Ashton, B., Middleton, J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 25, 2739-2749 (2007).
    4. Abdallah, B. M., Kassem, M. Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther. 15, 109-116 (2008).
    5. Erica, L., Herzog, L. C., Diane, S. Krause Plasticity of marrow-derived stem cells. Blood. 102, 3483-3493 (2003).
    6. Colter, D. C., Sekiya, I., Prockop, D. J. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A. 98, 7841-7845 (2001).

    Comments

    38 Comments

    hi,

    That is a really good explanation about how to derive the mesenchymal stem cells from rats. My work involves derivation of mesenchymal stem cells from mice. I tried to derive them in a similar way but was unsuccessful as my colonies dŒs not have any spindle shaped or round shaped as shown above. So, I would like to ask you can i implement the same kind of steps that are shown above for derivation of MSCs?? Further, how often do i have to change the medium once I plate the primary mesenchymal stem cells derived from the limbs?? You have not mentioned the percentage of trypsin u have used for deattachment of MSCs from the plastic plates?? I have used around 0.²5% trypsin with 5mins of incubation at 37C. But still the cells are not deattaching from the plates.
    Hope to get a reply soon from you.

    regards,
    Chandra Sekhar Amara
    Ph.D. Student
    Hartmann Lab,
    IMP, Vienna, Austria
    Reply

    Posted by: AnonymousMarch 29, 2010, 5:38 PM

    Hi Chandra,

    I haven't worked with mice MSCs and am not very sure whether you can get cells of the same shapes as rat MSCs. But you can try the experimental steps as listed. I usually keep ² dishes for every fresh isolation. Then I change media one day after isolation for one dish, and the other dish I change media two days after isolation. Some times change media one day after is better and some times change media two days after is better. After I pick the better dish, then I usually change media every 3 days.

    I used 0.²5% trypsin plus EDTA. Only trypsin itself dŒs not do a good job in lifting the cells. Supplement EDTA makes cell detachment much faster (usually takes 3~4 minutes for rMSCs I used). The trysin-EDTA I used is from invitrogen with catalogue number ²5²00-056.

    Good luck,

    Linxia
    Reply

    Posted by: AnonymousMarch 29, 2010, 9:00 PM

    Hi Linxia,

    I very much enjoyed your explanation in the vedio. I would like to know after you enrich the MSCs, can you stock them for later use and how many times (passage) i can use from one preparation of MSCs?

    best regards,
    Ava GUO
    Ph.D student
    Hong Kong, China
    Reply

    Posted by: AnonymousJune 23, 2010, 11:57 PM

    Hi Ava,

    Yes, you can freeze the cells in liquid nitrogen and use them later. I usually use the cells within ²0 passages.

    Thanks,
    Linxia
    Reply

    Posted by: AnonymousJune 24, 2010, 9:50 AM

    Hi Linxia,
    I am from china, i guess so are you. lol. I cannot get access to this video. Would you please send me a copy to jianhua_lee@1²6.com?
    Thanks a lot!!
    Lijianhua

    Reply

    Posted by: AnonymousOctober 9, 2011, 11:19 PM

    Hi Jianhua,

    Unfortunately we don't have a video copy either. If you don't access to the written up protocol either, I can send you a copy of that. Sorry couldn't help with the video.

    Linxia
    Reply

    Posted by: AnonymousOctober 10, 2011, 10:02 AM

    Hi linxia
    This is JŒ from montreal, would you like to send aslo a copy of protocols to my email address jŒchaochine@gmail.com. and also if possible some pictures of normal rat MSC, so i can compare their shapes with my cells.
    Reply

    Posted by: AnonymousOctober 27, 2011, 10:32 PM

    Hi JŒ,

    Sure. I will send you a copy of the protocol. As for the pictures, could you check out the figures in the protocol first and see if those are what you are looking for. If you need more, I am willing to send you some other figures we have.

    Linxia
    Reply

    Posted by: AnonymousOctober 28, 2011, 10:49 AM

    Dear Linxia
    I am Kinga from Budapest, Hungary. Could you please send me a copy of your protocol, I am starting my work with rat mesenchymal stem cells now and it would be really helpful.
    lakatoskinga87@gmail.com
    thank you very much
    Kinga Lakatos
    Reply

    Posted by: AnonymousNovember 16, 2011, 4:34 AM

    Hi Kinga,

    Sure, I will send you a copy of the protocol.

    Good luck,
    Linxia
    Reply

    Posted by: AnonymousNovember 16, 2011, 9:47 AM

    Hi LinXia,
    It is such a good video about how to derive the mesenchymal stem cells from rats and seperate the single-colony MSCs. I derive MSCs as shown above. But the cells are not deattaching from the ²5 flask with trypsin plus EDTA which is purchased from invitrogen with catalogue number ²5²00-056 as you describe. If there is any other reason, could you tell me?
    Looking forwad to getting a reply soon from you.

    best regards,
    Lijie Huang
    Ph.D. Student
    Wenzhou Medical College
    Wenzhou, Zhejiang, China
    Reply

    Posted by: AnonymousDecember 13, 2011, 1:00 AM

    Hi Lijie,

    If the cells are not lifting from the surface, try to incubate in Trypsin-EDTA for a little bit longer. After 5min, you can gentlly shake the flask to see whether the cells are coming up. If not, give them a couple of more minutes. Also make sure the Trypsin you get has EDTA in it. If it is only Trypsin (without EDTA), it takes much longer for the cells to deattach. Hope it helps.

    Good luck,
    Linxia
    Reply

    Posted by: AnonymousDecember 13, 2011, 11:55 AM

    Dear Linxia,

    I really need mouse bone marrow-derived messenchymal stem cell for my research but i haven't isolated them yet.
    Although your protocol is used for isolation rat bone marrow-MSC, i think i can use for my work. So can you send me a copy of your protocol to my email address: ntknguyen@hcmus.edu.vn
    Looking forwad to getting a reply soon from you.

    Nguyen Thi Kim Nguyen
    Laboratory of Stem Cell Research & Application
    Viet Nam National university of scinece, Viet Nam
    Reply

    Posted by: Nguyen Thi K.January 7, 2012, 10:02 AM

    Hi Nguyen,

    Just send you a copy of the protocol. Hope it helps.

    Linxia
    Reply

    Posted by: AnonymousJanuary 7, 2012, 10:16 AM

    Hi Linxia,

    I really enjoyed the video. I have isolated cell mesenchymal from murine bone marrow, but cultures do not proliferate (only until the second passages), I have used low-glucose DMEM plus 10% fetal bovine serum and Pen-Strep. Could you help me?


    Thanks,
    Reply

    Posted by: AnonymousJanuary 16, 2012, 11:44 AM

    Hi there,

    I have also experienced bad isolations. Some times the cells proliferate very slowly, show up as very large and flat morphology. These are senescent cells and is very unlikely to make them regrow healthy and fast. When this happens, I would do another isolation to get new cells and usually would get rid of the problem.
    Another thing is don't use medium that's been sitting in the fridge for very long. The pH changes over time and that affect the growth of the cells.

    Hope it helps,

    Linxia
    Reply

    Posted by: AnonymousJanuary 16, 2012, 1:50 PM

    Hi Linxia,
    I cannot get access to this video. I am very interested about MSC subpopulation. What can you tell me about this?Is it possible to have the protocol to separate single-colony derived cells using cloning cylinders ?
    Thank you very much
    mariarosaria
    Reply

    Posted by: AnonymousJanuary 18, 2012, 10:09 AM

    Hi Mariarosaria,

    Just let me know your email address and I can send you a copy of the protocol, which contains isolation of subpopulations.

    Thanks,
    Linxia

    Linxia
    Reply

    Posted by: AnonymousJanuary 18, 2012, 10:27 AM

    Hi Lixia,
    I dont have access to this video in JOVE. Please send me your protocol to my mail id. My mail id is msvnathan@gmail.com.
    Thanking you,
    S. Vaithinathan
    Reply

    Posted by: AnonymousJanuary 18, 2012, 2:40 PM

    Hi Vaithinathan,

    It's sent. Please check your email.

    Linxia
    Reply

    Posted by: AnonymousJanuary 18, 2012, 3:01 PM

    Dear Linxia

    I would like to know whether you add additional growth factors to the medium so to increase the confluency of the cells as i have carried out the experiment but the cells were quite few in number and never increased in number after a certain period of time (after 10 days).

    please kindly provide me the essential steps and precautions used during culturing and the factors required to increase the cell confluency.

    I have used high-glucose DMEM plus 15% fetal bovine serum and Pen-Strep. Could you help me and provide the composition and stimulating factors required for growth

    Thank
    Reply

    Posted by: AnonymousJanuary 18, 2012, 2:46 PM

    Hi Meera,

    I used low-glucose DMEM plus 10% FBS and pen-strep. No additional growth factors were added to the culture medium. The percentage of MSCs from bone marrow is very low, so for the first passage, you won't see many cells. But after a while, the adherent MSCs should grow and populate very fast and form colonies. If your cell number is not enough and they also don't grow fast enough, it might be related to the isolation. I have also experienced bad isolations. This seem to be also related to the animal that's used. When I use another animal, the problem is usually solved. You may try a new isolation. Also, you can optimize your culture medium. I haven't used high-glucose DMEM so I am not sure whether that would affect the growth of the cells. You could try low-glucose DMEM and also different concentration of FBS to see whether it helps.

    Linxia
    Reply

    Posted by: AnonymousJanuary 18, 2012, 3:09 PM

    Dear Linxia

    Thanks for your reply. I want to just discuss the protocol which i follow for the isolation of MSCs from Swiss albino mouse. I dissect out the femur and tibia, which is placed in DMEM (with L-glutamine, 4.5 gm glucose per litre with sodium pyruvate) with Pen/Strep soln in ice cold condition. This is placed as such for half an hour as i would be removing the tissues so to get bones only during flushing. While flushing I transfer the bone segments into Pen/Strp soln and one by one I flush the bone marrow cells with DMEM supplemented with 15 % FBS. When everything has been flushed out with the help of the syringe and the bones have become pale in colour. I centrifuge the whole cells and plate the pellets in DMEM supplemented with FBS. I use a primitive technique for isolation of MSC by plastic adherence. I change this media in 3 hours, maximum cells are aspirated out (HSCs). Then next day firstly I remove the media and allow the cells to grow with removal of media every day.

    Thus by this protocol I get cells which are adherent. I am unable to get these cells into confluent stage, I have studied that certain mesenchymal stimulating factors are available which are added to attain confluency. I studied ur papers abstract but it also mention no such use.

    Could u tell me the concentration of glucose in low-glucose DMEM.

    As I dont have access to this video in JOVE. Please send this protocol to my mail id and please do help me out in this context.

    Thank you so much in advance waiting for ur reply
    Reply

    Posted by: AnonymousJanuary 19, 2012, 12:40 AM

    Hi Meera,

    The DMEM I used is from Invitrogen (Cat # 11885-084). The complete formulation can be found from this website: http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/media_formulation.48.html.
    I see that you change medium 3 hours after plating your cells. Next time when you plating the cells, I would suggest that you plate several dishes. For dish 1, change medium as you normally do (say 3h after plating, or 6h later); for dish ², change medium the next day; for dish 3, change medium ² days later. From my experience, some times change medium the next day gives me better results while other times change medium ² days later gives me better results. I have not tried change medium 3 hours after plating, since I want to give the cells enough time to attach. You could try this out to see whether it may help.

    Also, you are isolating MSCs from mouse, which is also different from isolating from rat. I haven't done isolation from mouse before, so I am not sure if there are other critical steps involved. Maybe you could also look into some literature that particularly describes mouse MSC isolation.

    BTW, I still couldn't see you email address. You can drop me an email at zhanglin@msu.edu and I can send you a copy of the protocol.


    Good luck,
    Linxia
    Reply

    Posted by: AnonymousJanuary 19, 2012, 10:48 AM

    Dear Linxia,

    I am sorry couldn't get ur mail, Please send me your protocol to my mail id. My mail id is meera_nair30@rediffmail.com

    Thanking you,

    Meera
    Reply

    Posted by: AnonymousFebruary 4, 2012, 4:54 AM

    Hi Meera,

    The protocol is sent. Thanks, Linxia
    Reply

    Posted by: AnonymousFebruary 4, 2012, 10:27 AM

    Dear Linxia,
    I wish to know what is the freezing media you are using?
    and if you may, could you tell me the freezing procedure?
    Sincerely,
    Naama
    Reply

    Posted by: AnonymousFebruary 9, 2012, 2:44 AM

    Hi Naama,

    The cryopreservative medium I used is ²0% FBS and ²0% DMSO in MSC culture medium (But the final concentration is 10% FBS and 10% DMSO, please read the following text for why). Here is how I do it: First trypsinize MSCs, then add culture medium to stop trypsinization and collect cells. Spin down the cells and get rid of the supernatant medium. Resuspend cells in MSC culture medium, then add an equal amount of cryopreservative medium slowly and drop-wise. Mix quickly and gently (Now DMSO is 10%). Place cells in vials, put cells at -²0oC for a couple of hours, then put cells at -80oC overnight. The next day, store cells in liquid N². Hope it helps.

    Linxia
    Reply

    Posted by: AnonymousFebruary 9, 2012, 7:18 PM

    Hi, Thanks!!!!
    Reply

    Posted by: AnonymousFebruary 12, 2012, 1:59 AM

    Dear Linxia Zhang,

    Would you send to me your protocol?
    my email is Tung.biotech@gmail.com
    Thank you so much!

    Nguyen Thanh Tung
    Reply

    Posted by: Tung N.June 25, 2012, 3:35 AM

    Hi Nguyen, protocol has been sent. Thanks, Linxia
    Reply

    Posted by: AnonymousJune 25, 2012, 9:34 AM

    Dear Linxia
    I'm also working with rat MSC. I was wondering how you managed to get a good single cell suspension for FACS analysis because my cells like to attach to each other.
    And do you use any kind of positive control for you CD45- cells and isotypes?
    Thank you,
    Fabian
    Reply

    Posted by: Fabian L.January 28, 2013, 8:16 AM

    Hi Fabian,

    Usually my single cell suspension is pretty good. If you keep having problem, you could try use a 50um cell strainer to get rid of cell clumps. The MSCs we get are CD45-CD54+CD90+. I use magnetic cell sorting, flow cytometry is just used to verify that these cells dŒs express CD54 and CD90, but not CD45. I don't think you need a positive control for CD45- and isotypes here. The peaks for the CD45- and isotypes basically overlaps with the 'cells only' control, which dŒs not have fluorescence.

    Thanks,
    Linxia
    Reply

    Posted by: AnonymousJanuary 28, 2013, 10:44 AM

    Hi Linxia,

    thank you for the detailed video. I'm working on RCS rat and isolate MSC from it. I've followed your protocol but got CD45+/CD54+/CD90+ cell population. Would longer incubation (almost ² weeks for each incubation) cause the problem? Would higher concentration of Ab cause the false results? Other than that, I don't know what should be pay attention. Do you have any suggestions? Thanks!

    yuchun
    Reply

    Posted by: yuchun t.April 11, 2013, 8:33 PM

    Hi Yuchun,

    When you say "almost ² weeks for each incubation", do you mean you incubate the cells with the primary antibody for ~² weeks? I only incubate with the primary antibody for ~30min on ice. Also, you probably need to optimize the antibody concentration. Try a couple of dilutions and pick the best one from your FACS results, then stick to that concentration. Hope it helps.

    Thanks,
    Linxia
    Reply

    Posted by: Linxia Z.April 15, 2013, 10:22 AM

    Hi Linxia,

    thanks for your answer. No, the cells are harvested for ² weeks (1st: isolation from femurs and tibias, ²nd: after enrichment iwth CD54, and CD90). For primary antibody I incubate also 30min on ice. It seems like the selection (CD54 & CD90) dŒsn't work. Or other possibilities? Thanks a lot!

    yuchun

    Reply

    Posted by: yuchun t.April 15, 2013, 12:27 PM

    Hi Yuchun,

    So you get CD45+/CD54+/CD90+ cells? Do you do cell sorting before verify with FACS? What you could do is to sort out CD45- cells, then sort out CD54+/CD90+ cells from the CD45- population. Also, make sure your CD45 antibody is good. Some polyclonal antibodies may have lots of non-specific binding. It may recognize other antigens and therefore give you a positive signal. You may want to try with another antibody to see if it fixes the problem.

    Linxia
    Reply

    Posted by: Linxia Z.April 15, 2013, 12:41 PM

    Hi Linxia,

    I would be really very thankful if you have some time to forward the protocol to mariam.foad@gmail.com,

    Thank you,
    Mariam
    Reply

    Posted by: mariam f.February 13, 2014, 8:54 AM

    Post a Question / Comment / Request

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

    Metrics

    Waiting
    simple hit counter