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 JoVE Biology

Preparing T Cell Growth Factor from Rat Splenocytes

1, 1

1Department of Physiology and Biophysics, University of California, Irvine (UCI)

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    Summary

    We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

    Date Published: 10/31/2007, Issue 10; doi: 10.3791/402

    Cite this Article

    Beeton, C., Chandy, K. G. Preparing T Cell Growth Factor from Rat Splenocytes. J. Vis. Exp. (10), e402, doi:10.3791/402 (2007).

    Abstract

    Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2.

    Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20°C.

    Protocol

    1. Take Lewis rat spleens (rats between 160-200g are best). Dilacerate on ice in a petri dish containing PBS + antibiotics (PBS-PS) using a cell strainer. Put in a 50 ml tube. Fill with PBS-PS.
    2. Spin for 10 min at 4°C to pellet the cells.
    3. Wash the cells twice.
    4. Resuspend the pellet in NH4Cl 0.15 M (5 ml per spleen). Mix gently and continuously with a pipet for 3 min on ice to lyse the erythrocytes. Fill the tube with medium.
    5. Spin for 10 min at 4°C to pellet the cells.
    6. Wash the cells twice.
    7. Count the cells. A rat spleen gives 200-250 million cells.
    8. Seed the cells at 2 million per ml in complete medium: 50 ml per 75 cm2 flask.
    9. Let grow for 48 hours in the incubator.
    10. Spin 15 min at 4°C to pellet the cells.
    11. Collect the supernatant; discard the cells.
    12. Add 15 mg/ml a methyl mannoside to the supernatant Mix thoroughly.
    13. Filter (0.2 mm)
    14. Aliquot and store at -20°C Can be kept at 4°C for 10 days if necessary.

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    Discussion

    We prepare TCGF from Lewis rat splenocytes since we regularly euthanize naive rats from this strain to harvest serum and thymi to stimulate Lewis rat T cell lines in vitro. This TCGF can be used to promote the growth and survival of T cell lines from other rat strains. TCGF can also be prepared from other strains of rats.

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    Disclosures

    The authors have nothing to disclose.

    Materials

    Name Type Company Catalog Number Comments
    PBS, 1x, sterile Reagent GIBCO, by Life Technologies 14040-182
    Penicillin / Streptomycin 100x Reagent Sigma-Aldrich P0781 Add 5 ml of 100x solution to 500 ml PBS to prepare PBS-PS
    Cell strainer, 70 um diameter Tool Fisher Scientific 08-771-2
    Ammonium Chloride (NH4Cl) Reagent Sigma-Aldrich A0171 Prepare a 0.15 M solution in sterile distilled water, keep cold
    RPMI 1640 Reagent GIBCO, by Life Technologies 21870-092
    Fetal Bovine Serum (FCS, FBS) Reagent GIBCO, by Life Technologies 16140-071 Heat-inactivated
    Penicillin / Streptomycin / L-Glutamine Reagent Cambrex/BioWhittaker 17-718R PSG
    RPMI vitamins, 100x Reagent Sigma-Aldrich R7256
    Sodium pyruvate, 100x Reagent Sigma-Aldrich S8636
    Non-essential amino acids, 100x Reagent Sigma-Aldrich
    Beta-mercapt–thanol Reagent Sigma-Aldrich M7522
    alpha methyl mannoside Reagent Sigma-Aldrich M6882
    Concanavalin A Reagent Sigma-Aldrich M6882
    To prepare complete culture medium add the following to a 500 ml bottle of RPMI 1640 and sterile-filter: 10% FCS; 1 bottle of PSG; 5 ml RPMI vitamins; 5 ml sodium pyruvate; 5 ml non-essential amino acids; 50 uM beta-mercapt–thanol; 2 ug/ml Concanavalin A.

    References

    1. Beeton C., Barbaria J., Devaux J., Benoliel A.-M., Gola M., Sabatier J.-M., Bernard D., Crest M., Beraud E. Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation. J. Immunol. 166:936-944 (2001).

    2. Beeton C., Wulff H., Barbaria J., Clot-Faybesse O., Pennington M., Bernard D., Cahalan M.D., Chandy K.G., Beraud E. Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc. Natl. Acad. Sci. USA. 98:13942-13947 (2001).

    3. Mor, F., Cohen, I.R. Propagation of Lewis rat encephalitogenic T cell lines: T-cell-growth-factor is superior to recombinant IL-2. J. Neuroimmunol. 123: 76-82 (2002).

    Comments

    37 Comments

    Thank you for a very interesting and useful protocol. I would be interesting in knowing if the cells that are pelleted after 48hours and discarded - are these considered T cells at this time and can these be subsequently expanded ?
    Reply

    Posted by: AnonymousJune 16, 2008, 5:40 PM

    Most of these cells are indeed activated T cells but this is not a pure population, I would recommend checking the % of CD3+ cells to know exactly what you have in your mix. If you want a clean T cell population you may need to do a separation by either magnetic beads or flow cytometry. These T cells can be expanded after the 48-hr culture. I would recommend resuspending the pellet in fresh medium (same as described above) with no ConA but with 10% TCGF and keeping them in culture for 4-7 days before re-stimulating them with ConA (or any stimulus you want to use). T cells will divide a lot in the presence of TCGF so you will want to watch them and split them as they grow. You will probably need to split them every ² days. The re-stimulation will work best if you give the T cells some irradiated antigen-presenting cells but you should get some stimulation with ConA alone and no antigen-presenting cells. During the first stimulation described above, non-T cell splenocytes will act as antigen-presenting cells but will be dead and useless at the time of re-stimulation. Each round of stimulation will increase your T cell %. I hope this helps and good luck with your experiments!
    Reply

    Posted by: Christine B.June 16, 2008, 6:48 PM

    Thanks for the previous response - I wanted to know a couple more things if possible - after 10 - 15 ml aliquots are made and placed in -²0 : (1) how much do you tend to use at a time ? (²) are there any porblems with the sample thawing and then being refrozen?
    Reply

    Posted by: AnonymousJune 27, 2008, 10:52 AM

    We use the TCGF at 10% when growing T cells in the expansion phase so we use anything from 5 ml to 50 ml of TCGF on a given day. I have never re-frozen thawed TCGF and would not recommend it but you can keep the thawed tube in the fridge for 10 days, this works well.
    Reply

    Posted by: Christine B.June 30, 2008, 10:33 AM

    HI Christine, Thanks for your help in answering some of the quesitons I have had. I have some more for you ! I have used the TCGF to expand a separate population of T cells. What I wanted to ask was (a) when you are splitting the cells - what is your cell count approx -eg do you split if the cell count is greater than 1 x10^6 /ml (b) in terms of the irradiated APC - what did you use and how did you go about doing this? (c) if I was to use ConA alone to stimulate the cell line - would I need to use the methyl mannoside again to deactivate it after 48hours ? Thanks for any  help you may be able to give Rohit 
    Reply

    Posted by: AnonymousAugust 4, 2008, 6:10 PM

    Hi Rohit, Sorry for this long delay in ansering your questions. (a) I seed the cells at 0.² million/ml and usually need to split them after a couple of days, when they reach 0.6 - 0.8 million/ml. I try to never let them go over 1 million/ml. (b) for rat T cell activation I use irradiated rat thymocytes (see the first steps of this video: Beeton C., Chandy K.G. (²007) Induction and monitoring of adoptive delayed type hypersensitivity in rats, J. Vis.  Exp. 8, http://www.jove.com/index/Details.stp?ID=3²5)   For human T cells I use irradiated mononuclear cells (or purified B cells) from the same donor.   (c) No, you won't need to use the MM because in that case you are interested in the cells and not the supernatant, so you would throw the excess ConA away with the supernatant.   Christine
    Reply

    Posted by: AnonymousAugust 15, 2008, 4:43 PM

    Dear Christine, I have got a question regarding the red blood cell lysis buffer you have used. I found in other protocols that they used NH4Cl in addition EDTA and KHCO3. Could you please let me know why you have just used NH4Cl. I am doing Lewis rat spleen culture for the first time and I am concerned about this red blood cell lysis step. Do you think there is a possibility that the red blood cells will cluster if not treated with EDTA. Another question is regarding the cell strainer you have used (70u). I was planning to use a 40u cell strainer to avoid having cell clusters. From your experiance, please let me know about using a 40n cell strainer.   Regards, Karima
    Reply

    Posted by: AnonymousSeptember 11, 2008, 11:27 AM

    Since we are interested in the supernatant and not the cells we have never really been worried about cells clumping. We haven't observed any significant clumping during this procedure but you can add EDTA to your solution, it will not drastically affect its osmolarity. All the splenocytes are less than 15 um in diameter so a 40 um cell strainer will give you good results. Regards, Christine
    Reply

    Posted by: Christine B.October 2, 2008, 1:14 PM

    What are the difference between Heat activated and non heat inactivated Fetal calf serum (FCS)? Please explain with examples.
    Reply

    Posted by: AnonymousMarch 14, 2009, 10:23 PM

    Heat inactivation (56C for 30 min) of serum is done to degrade the complement that could induce cell lyzis in the presence of antibodies. This procedure was established several decades ago. Heat inactivation may not be necessary, depending on the cell grown and the source/technique of serum production. I have never tried using non heat-inactivated serum as I don't want to change what is not broken and this has always worked well for us. If you are starting a new protocol I would certainly encourage you to test a sample of the same serum, before and after heat inactivation to decide on which works best for you.
    Reply

    Posted by: Christine B.March 17, 2009, 7:03 PM

    Heat inactivation (56C for 30 min) of serum is done to degrade the complement that could induce cell lyzis in the presence of antibodies. This procedure was established several decades ago. Heat inactivation may not be necessary, depending on the cell grown and the source/technique of serum production. I have never tried using non heat-inactivated serum as I don't want to change what is not broken and this has always worked well for us. If you are starting a new protocol I would certainly encourage you to test a sample of the same serum, before and after heat inactivation to decide on which works best for you.
    Reply

    Posted by: Christine B.March 17, 2009, 7:04 PM

    Rat T-stim that we normally order from BD dŒsn't recommend freezing, but rather storing at 4'C. I would think freezing it would possibly increase the half-life whereas keeping at 4'C it would go bad within a matter of weeks. Do you think there's a big issue freezing and thawing, and why dŒs BD not recommend it? Thanks
    Reply

    Posted by: AnonymousMarch 16, 2009, 4:41 PM

    I don’t know why BD would not recommend storing TCGF at -²0C. I have never used TCGF other that “home-made” as we always euthanized healthy rats with no other use for their spleen. We have been very successful in storing this TCGF at -²0C for several months but found that it lost efficacy if kept for more than 10-15 days at 4C. We try to aliquot the new TCGF before freezing but have also frozen it bulk (500 ml at a time) and then thawed, aliquoted and re-froze without loosing activity. I have never tried several cycles of freeze-thaw though and we try to prepare aliquots that can be used up within days of thawing.
    Reply

    Posted by: Christine B.March 17, 2009, 7:06 PM

    There is an error in the catalog number for Concanavalin A. It should read C041² (from Sigma).
    Reply

    Posted by: AnonymousJune 15, 2009, 1:34 PM

    Hi Christine. Thanks for the video, very informative. I was wondering if you knew about repeating the same procedure but with mice instead of rats? Or using the TCGF obtained from rats for maintaining a mouse T cell line?
    Reply

    Posted by: AnonymousFebruary 10, 2010, 6:50 PM

    Hi Dean, I have never tried preparing TCGF from mouse spleens so I don't know for sure that it would work but I can't think of any reason why it shouldn't. Concanavalin A activates mouse T cells so that shouldn't be a problem. The only thing is mouse spleens will be way smaller than rat spleens so you would need many more mice to generate the same amount of TCGF.
    I have never tried rat TCGF for growing mouse cells but I have used rat TCGF for growing human T cells and it works very well. So I would definitely recommend trying rat TCGF on mouse T cells.
    Hope this helps!
    Reply

    Posted by: AnonymousFebruary 10, 2010, 6:55 PM

    Hi Chistine,
    I'm trying to produce cytokines(TNFalpha, IL-², IL-4 IL-10, TGF-b and IFNgama) by stimulated PBMC. I isolated PBMC from whole blood by Ficoll method. In 6 well tissue culture plate, I added 10 ug/mL PHA_L (sigma) for 105cells/ml and incubated 3 days. I used cell culture supernatants for ELISA assay but cytokine levels were very low. Can yu give me some advice?
    Thanks,
    Tuba
    Reply

    Posted by: AnonymousApril 22, 2010, 3:54 AM

    Hi Tuba, Thank you for watching this video.
    If you are interested in the cytokines and not the cells I would use the cells at a higher density (² million/ml). The medium should change color by the time you harvest the supernatants (golden color).
    You also want to make sure the Ficoll is well washed off the cells before plating them for culture.
    I hope this helps!
    Christine
    Reply

    Posted by: AnonymousApril 22, 2010, 5:57 PM

    Hi Chistine,
    Thank you for your video. It's very useful. I have two question,
    1) why thymocytes can be APC after irradiation? Can I use thymocytes as APC, If thymocytes aren't irradiated?
    ²) I know you use the TCGF to culture the T cell clone, and I will do the same thing. Can you give me detail protocols? My email is ammszdy@1²6.com.
    Your reply will be highly appreciated.
    Reply

    Posted by: AnonymousJune 24, 2010, 2:40 AM

    1) The thymocytes must be irradiated to prevent them from growing. Irradiation will allow them to remain alive for a few hours to process and present antigens to the T cells but they can't divide and will die. Otherwise they would grow as fast (or faster) as the T cells and overtake the medium. And then you wouldn't have a clean population any more.
    ²) The stimulation of T cell lines (specific to ovalbumin) is shown in this JoVE video: Beeton C., Chandy K.G. (²007) Induction and monitoring of adoptive delayed type hypersensitivity in rats, J. Vis. Exp. 8, www.jove.com/index/Details.stp?ID=3²5
    If you want to keep the cells in culture, two days after stimulation you need to change the medium to DMEM + 10% FBS + 10% TCGF and seed the cells at 0.² million/ml. Add medium as needed over the next 4-6 days to keep the cell density below 1 million/ml (0.² - 0.5 million/ml is best). After 4-6 days the cells will need to be stimulated again as in the JoVE video above.
    Hope this helps.
    Reply

    Posted by: AnonymousJune 24, 2010, 10:47 AM

    HI Christine,
    Can I use Mitomycin C-treated splenocytes as APC to stimulate T cell line?
    Reply

    Posted by: AnonymousJuly 11, 2010, 9:52 AM

    You can, it has the same final effect on the APCs as irradiation. You however have to be extremely careful at washing your mitomycin C-treated APCs before mising them to the T cells. I don't really like using this technique and would recommend using irradiation if at all possible. Even if you wash your APCs really really well, when they die I am not sure if they won't release a little mitomycin C and that could affect your T cells. Especially if you do that repeatedly to keep a line/clone in culture.
    BTW, the typical dose is 50 ug/ml of mitomycin C.
    Reply

    Posted by: AnonymousJuly 21, 2010, 5:55 PM

    This may seem like a very simple question, but how do people typically irradiate splenocytes? Do you need special apparatus?
    Reply

    Posted by: AnonymousMay 3, 2011, 7:18 PM

    Yes, you do need a special apparatus called an irradiator. It contains a very small radioactive source that you expose your sample to.
    If you do not have access to an irradiator, you can use mitomycin C instead of irradiation, but check my previous comment above about precautions to take when using this reagent.
    Reply

    Posted by: AnonymousMay 3, 2011, 10:12 PM

    Hi christine, actually you have a very very amazing and useful video, i got a lot of information in this field and how we can deal with rats and spleen harveting procedure.

    Thank you agian christine
    Reply

    Posted by: yaseen a.September 6, 2011, 5:25 AM

    Hi Christine and everybody,

    thanks a lot for the video. I need to prepare ConA activated medium of splenocytes and your protocol will be very very helpful!! I am a student and have nearly no experiences in cell culture, and completely none in immunology. How can I, afterwards, show that the cytokines really are in the supernatant? I think I should apply an ELISA but I don´t know which antibodies to coat in the wells. Could anybody help me with that, please? ( I won´t be able to purchase a whole ELISA-Kit for this experiment) Any advice would help me. Thanks in advance.
    Reply

    Posted by: AnonymousSeptember 30, 2011, 9:28 AM

    This supernatant will contain a mix of a large number of cytokines. Some at high concentrations, some at low concentrations, and some in minute amounts. Your best bet is to look for IL-², which will be the major cytokine in the mix.
    You have several options: use an ELISA (or IL-² beads by flow cytometry) to detect secreted IL-² in the supernatant, use intracellular staining for IL-² in the splenocytes by flow cytometry, or use your supernatant to grow IL-² dependent cells (such as CTL-L² cells or NK-9² cells, both cell lines are pretty widely available and at least one can be purchased from the ATCC).
    It never hurts to run controls, especially when you produce this supernatant for the first time, but if it turns the color of gold at the end of the culture time, you are almost sure it has worked and will give you good results.
    Good luck!
    Christine
    Reply

    Posted by: AnonymousSeptember 30, 2011, 11:24 PM

    Dear Dr Beeton
    Since I couldn't be registered, could I please you to send me this video and the related article. Thank you so much.
    Regards
    Reply

    Posted by: AnonymousDecember 18, 2011, 5:49 AM

    JoVE holds the copyright for this video-article and we cannot share it without their written approval.
    Please communicate directly with members of the JoVE team.
    Reply

    Posted by: AnonymousDecember 19, 2011, 4:35 PM

    Hi Christine,
    I am about to do a re-stimulation culture with rat lymph node cells and I was wondering if I could use medium with FCS rather than the recommended autologous rat serum? Thanky you.
    Best regards
    Reply

    Posted by: Lukasz S.August 16, 2012, 8:34 AM

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