Preparing T Cell Growth Factor from Rat Splenocytes

Christine Beeton; K. George Chandy

doi:10.3791/402

Cite this Article: Preparing T Cell Growth Factor from Rat Splenocytes

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

Abstract: Preparing T Cell Growth Factor from Rat Splenocytes

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: Preparing T Cell Growth Factor from Rat Splenocytes

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.
Spin for 10 min at 4°C to pellet the cells.
Wash the cells twice.
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.
Spin for 10 min at 4°C to pellet the cells.
Wash the cells twice.
Count the cells. A rat spleen gives 200-250 million cells.
Seed the cells at 2 million per ml in complete medium: 50 ml per 75 cm² flask.
Let grow for 48 hours in the incubator.
Spin 15 min at 4°C to pellet the cells.
Collect the supernatant; discard the cells.
Add 15 mg/ml a methyl mannoside to the supernatant. Mix thoroughly.
Filter (0.2 mm)
Aliquot and store at -20°C. Can be kept at 4°C for 10 days if necessary.

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Discussion: Preparing T Cell Growth Factor from Rat Splenocytes

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: Preparing T Cell Growth Factor from Rat Splenocytes

The authors have nothing to disclose.

Materials: Preparing T Cell Growth Factor from Rat Splenocytes

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-mercaptoethanol	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-mercaptoethanol; 2 ug/ml Concanavalin A.

References: Preparing T Cell Growth Factor from Rat Splenocytes

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).

Ask the Author: Preparing T Cell Growth Factor from Rat Splenocytes

17 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 ?

Posted by: Rohit SethJune 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!

1.1

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

Thanks for your response. Thats very helpful

1.1.1

Posted by: AnonymousJune 25, 2008, 6:37 AM

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?

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.

2.1

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

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

3.1

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

Hi Christine

Thanks for the reply! thats really helpful.

regards

rohit

3.1.1

Posted by: AnonymousAugust 26, 2008, 6:47 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

Posted by: KarimaSeptember 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

4.1

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.

Posted by: AnowerMarch 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.

5.1

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.

5.2

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

Rat T-stim that we normally order from BD doesn'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 does BD not recommend it? Thanks

Posted by: EdMarch 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.

6.1

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).

Posted by: ChristineJune 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?

Posted by: DeanFebruary 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!

8.1

Posted by: ChristineFebruary 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

Posted by: TubaApril 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

9.1

Posted by: ChristineApril 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.

Posted by: ammszdyJune 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.

10.1

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

Thank you very much!
I have another questions: that is, during the maintenance of T cell line whether irradiated APC is necessary? and do you induce T cell anergy? I am doing it, but the T cell line will die after the rest for 24h without any additional cytokine in medium culture. I am very confused.

10.1.1

Posted by: zhao d.June 26, 2010, 4:49 AM

When do your lymphocytes die? After 24 hours in medium + TCGF? That would mean your batch of TCGF is not a good one. TCGF is full of cytokines (including lots of IL-2) and using 10% should keep your cells alive. No need to add irradiated APCs when you add TCGF, their role is only to present antigen during the activation step.
Are you stimulating an established cell line or starting one from primary cells?
If starting from primary cells, I would expect the majority to die during the first rounds of in vitro activation because not all cells are specific to your antigen. It can be scary, but it is normal, just keep taking care of the few cells that remain alive and eventually you will have an antigen-specific line growing nicely.
If you are using an established cell line, how are you doing your expansion? I stimulate the cells for 2 days in medium + 1% rat serum (when using a rat cell line) + antigen + irradiated APCs. Then I switch to medium + 10% FCS + 10% TCGF (no APCs, no antigen) for 4-6 days, adding medium as needed every 2-3 days. This should not induce anergy.
What color is your TCGF? It should be gold. If it is pink, that means the splenocytes didn’t grow well and didn’t produce the cytokines you need in TCGF. If it is yellow, the splenocytes could have started dying, release “toxic waste”.
After the 48-hour stimulation, were your lymphocytes activated (i.e. enlarged and forming clumps of cells)? If the cells were small and all single, then the activation did not work and the cells will eventually die. In that case, check your ratio T cell/APCs and number of cells/volume of medium, check the level of irradiation of your APCs (you don’t want them to die too fast, before they can process and present the antigen), and check your antigen concentration.

10.1.1.1

Posted by: ChristineJune 26, 2010, 4:35 PM

Thank you.
I used the system of anti-CD3 and CD28 antibody to establish the mouse Th1 cell line not antigen-specific. Maybe it can not be called cell line. After one round of stimulation I used the cells to induce anergy induced with CD3 antibody alone, and the control of anergy is the Th1 cells resting in the culture medium without any cytokines or TCGF. After 24 hours, a lot of Th1 cells died in anergy or control of anergy condition. I don't konow why. I have been thinking whether my Th1 cells is suitable for this experiment. I have done it for a long time. It is very depressing.

10.1.1.1.1

Posted by: zhao d.June 26, 2010, 10:54 PM

I don't have any experience with anergy so i can't really help you there. I suggest conctating a lab that has extensive experience with inducing anergy and has published in the field in the recent years.

10.1.1.1.1.1

Posted by: ChristineJuly 21, 2010, 5:57 PM

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

Posted by: BuzzJuly 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.

11.1

Posted by: ChristineJuly 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?

Posted by: JaneMay 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.

12.1

Posted by: ChristineMay 3, 2011, 10:11 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

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.

Posted by: student_visitorSeptember 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

14.1

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

Thanks a lot for your help!

14.1.1

Posted by: Student_visitorOctober 4, 2011, 8:28 AM

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

Posted by: AmenehDecember 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.

Posted by: ChristineDecember 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

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

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Date Published	10/31/2007
JoVE Section	General
JoVE Issue	10
Comments	17 Comments
View Count	Loading... (Details…)
DOI	doi:10.3791/402