In Vitro Systems for the Study of T Cell Development: Fetal Thymus Organ Culture and OP9‐DL1 Cell Coculture

Fred Ramsdell1, Juan Carlos Zúñiga‐Pflücker2, Yousuke Takahama3

1 Immunex Corporation, Seattle, Washington, 2 Sunnybrook & Women's Research, Institute University of Toronto, Toronto, 3 University of Tokushima, Tokushima
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.18
DOI:  10.1002/0471142735.im0318s71
Online Posting Date:  March, 2006
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Most T cell development occurs within the thymus and includes a series of selection processes that are, in large part, still poorly understood. Studies of T cell development have been greatly advanced by the description of multiple phenotypic subsets of T cells and their maturational relationships. This unit describes a system for observing and modulating T cell development in vitro via the culture of entire mouse fetal thymic lobes. Methods are included for the isolation of fetal thymi and culture to allow for normal T cell development on either transwell plates or Gelfoam sponges. A method for depleting hematopoietic cells from thymic lobes using 2′‐deoxyguanosine and subsequent reconstitution with precursor cells is also described. This protocol is valuable for the study of tolerance and T cell selection. A support protocol describing methods of altering and monitoring T cell development are outlined. In addition, methods for culturing fetal thymic lobes under high oxygen submersion conditions and for the preparation of reaggregate thymus organ cultures are provided. Finally, a simple and practical method that allows for the thymus‐independent generation of T cells from defined sources of stem/progenitor cells by OP9‐DL1 coculture is described.

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Table of Contents

  • Basic Protocol 1: Isolation and Culture of Thymic Lobes on Gelfoam Sponges
  • Alternate Protocol 1: Culture of Fetal Thymic Lobes in Transwell Plates
  • Alternate Protocol 2: Hematopoietic Cell Depletion and Reconstruction of Fetal Thymic Lobes
  • Alternate Protocol 3: T Cell Development in High‐Oxygen Submersion Fetal Thymus Organ Culture
  • Alternate Protocol 4: T Cell Development in Reaggregate Thymus Organ Culture
  • Alternate Protocol 5: T Cell Development in OP9‐DL1 Cell Cocultures
  • Support Protocol 1: Monitoring T Cell Development and Selection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolation and Culture of Thymic Lobes on Gelfoam Sponges

  • Day‐14 fetal mice from timed, pregnant C57BL/6 females
  • Medium 199‐5 (see recipe), room temperature and ice cold
  • Complete DMEM‐10 medium ( appendix 2A)
  • Surgical scissors
  • Microdissecting tweezers with extra‐fine tips (Dumont no. 5 straight or no. 7 curved)
  • 15 × 100–mm and 12 × 75–mm petri dishes (e.g., Falcon)
  • 50 × 50–mm 4‐ply sterile gauze (BD Biosciences)
  • Dissecting microscope
  • Blunt‐end tweezers with extra‐fine tips (e.g., Perry tweezers or iris tweezers)
  • 0 × 60 × 7‐mm Gelfoam gelatin sponges (Upjohn)
  • 6‐well tissue culture plates (e.g., Costar, cat. no. 3506)
  • 0.8‐µm and 13‐mm round Nucleopore filters (Whatman)
  • 3‐ml and 1‐ml syringes
  • 70‐µm nylon‐mesh cell strainers (Nytex or Falcon)
  • 15‐ml conical polypropylene centrifuge tubes
  • Additional reagents and equipment for euthanasia of mice (unit 1.8) and determining cell viability by trypan blue exclusion ( appendix 3B)

Alternate Protocol 1: Culture of Fetal Thymic Lobes in Transwell Plates

  • Costar 6‐well transwell plates (Costar no. 3412; 0.4‐µm pore size)

Alternate Protocol 2: Hematopoietic Cell Depletion and Reconstruction of Fetal Thymic Lobes

  • 1.35 mM (36 mg/100 ml) 2′‐deoxyguanosine (2‐dG; Sigma) in complete DMEM‐10 (see appendix 2A for DMEM‐10), filter sterilized
  • Source of progenitor/stem cells: bone marrow of adult mouse, fetal liver, thymi from day‐13 to day‐14 fetal mice, or adult thymi
  • 5‐ml syringe with 25‐G needle
  • Terasaki plates (e.g., Nunc)
  • Additional reagents and equipment for preparation of single‐cell suspensions (unit 3.1), T cell depletion (unit 3.4), and flow cytometry and cell sorting (Chapter 5)

Alternate Protocol 3: T Cell Development in High‐Oxygen Submersion Fetal Thymus Organ Culture

  • Gas mixture consisting of 70% O 2, 25% N 2, and 5% CO 2 (can be custom ordered from most gas‐supply services)
  • 96‐well round‐bottom plates (Falcon)
  • Centrifuge with microtiter plate carrier
  • 3‐ to 5‐liter plastic bags
  • Plastic bag heat‐sealer

Alternate Protocol 4: T Cell Development in Reaggregate Thymus Organ Culture

  • Day‐15 fetal mice from timed, pregnant C57BL/6 females
  • Phosphate‐buffered saline (PBS; Ca2+‐ and Mg2+‐free; appendix 2A)
  • 0.5% trypsin/5.3 mM EDTA stock solution (e.g., Invitrogen)
  • 12 × 15–mm, 5‐ml sterile round‐bottom polypropylene snap‐cap tubes (e.g., Falcon)

Alternate Protocol 5: T Cell Development in OP9‐DL1 Cell Cocultures

  • OP9‐DL1 cells (OP9 cells; Riken cell repository,, retrovirally transduced to express the Notch ligand Delta‐like‐1; Schmitt and Zúñiga‐Pflücker, 2002)
  • Hematopoietic progenitors or stem cells of interest (unit 22.1)
  • 5 µg/ml (1000×) Flt‐3 ligand (e.g., R&D Systems cat. no. 308‐FK); store at −80°C
  • 1 µg/ml (1000×) IL‐7 (e.g., R&D Systems cat. no. 407‐ML); store at −80°C
  • 6‐well tissue culture plates
  • 70‐µm nylon cell strainers
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Literature Cited

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   Anderson, G., Jenkinson, E.J., Moore, N.C., and Owen, J.J.T. 1993. MHC class II‐positive epithelium and mesenchyme cells are both required for T‐cell development in the thymus. Nature 362:70‐73.
   Anderson, M.K., Weiss, A.H., Hernandez‐Hoyos, G., Dionne, C.J., and Rothenberg, E.V. 2002. Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro‐T cell stage. Immunity 16:285‐296.
   Ashton‐Rickardt, P.G., Bandeira, A., Delaney, J.R., Van Kaer, L., Pircher, H.P., Zinkernagel, R.M., and Tonegawa, S. 1994. Evidence for a differential avidity model of T cell selection in the thymus. Cell 76:651‐663.
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   Fine, J.S. and Kruisbeek, A.M. 1991. The role of LFA‐1/ICAM‐1 interactions during murine T lymphocyte development. J. Immunol. 147:2852‐2859.
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   Jenkinson, E.J., Franchi, L.L, Kingston, R., and Owen, J.J.T. 1982. Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: Application in the production of chimeric thymus rudiments. Eur. J. Immunol. 12:583‐587.
   Jenkinson, E.J., Anderson, G., and Owen J.J.T. 1992. Studies on T cell maturation on defined thymic stromal cell populations in vitro. J. Exp. Med. 176:845‐853.
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   Plum, J., De Smedt, M., Tison, B., and LeClercq, G. 1989. Influence of antibodies neutralizing cytokines on murine fetal thymic organ cultures. Thymus 13:83‐93.
   Plum, J., De Smedt, M., Billiau, A., Heremans, H., LeClercq, G., and Tison, B. 1991. IFNg reverses IL‐4 inhibition of fetal thymus growth in organ culture. J. Immunol. 147:50‐54.
   Ready, A.R., Jenkinson, E.J., Kingston, R., and Owen, J.J.T. 1984. Successful transplantation across major histocomplatibility barrier of deoxyguanosine‐treated embryonic thymus expressing class II antigens. Nature 310:231‐233.
   Schmitt, T.M. and Zúñiga‐Pflücker, J.C. 2002. Induction of T cell development from hematopoietic progenitor cells by Delta‐like‐1 in vitro. Immunity 17:749‐756.
   Schmitt, T.M., de Pooter, R.F., Gronski, M.A., Cho, S.K., Ohashi, P.S., and Zúñiga‐Pflücker, J.C. 2004. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat. Immunol. 5:410‐417.
   Sebzda, E., Wallace, V.A., Mayer, J., Yeung, R.S., Mak, T.W., and Ohashi, P.S. 1994. Positive and negative thymocyte selection induced by different concentrations of a single peptide. Science 263:1615‐1618.
   Skinner, M., Le Gros, G., Marbrook, J., and Watson, J.D. 1987. Development of fetal thymocytes in organ cultures: Effects of interleukin‐2. J. Exp. Med. 165:1481‐1493.
   Smith, C.A., Williams, G.T., Kingston, R., Jenkinson, E.J., and Owen, J.J.T. 1989. Antibodies to CD3/T‐cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337:181‐184.
   Sugawara, T., Di Bartolo, V., Miyazaki, T., Nakauchi, H., Acuto, O., and Takahama, Y. 1998. An improved retroviral transfer technique demonstrates inhibition of CD4‐CD8‐ thymocyte development by kinase‐inactive ZAP‐70. J. Immunol. 161:2888‐2894.
   Ueno, T., Liu, C., Nitta, T., and Takahama, Y. 2005. The development of T lymphocytes in mouse fetal thymus organ culture. Methods Mol Biol. 290:117‐134.
   Waanders, G.A., Godfrey, D.I., and Boyd, R.L. 1989. Moduation of T‐cell differentiation in murine fetal thymus organ cultures. Thymus 13:73‐82.
   Watanabe, Y. and Katsura, Y. 1993. Development of T cell receptor αβ‐bearing T cells in the submersion organ culture of murine fetal thymus at high oxygen concentration. Eur. J. Immunol. 23:200‐205.
   Watanabe, Y., Gyotoku, J.‐I., and Katsura, Y. 1989. Analysis of the development of T cells by transferring precursors into cultured fetal thymus with a microinjector. Thymus 13:57‐71.
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   Zúñiga‐Pflücker, J.C., Jones, L.A., Longo, D.L., and Kruisbeek, A.M. 1989. Both TCR/MHC and accessory molecule/MHC interactions are required for positive and negative selection of mature T cells in the thymus. Cold Spring Harb. Symp. Quant. Biol. 49:153‐158.
Key References
   Ceredig, R. 1988. Differentiation potential of 14‐day fetal mouse thymocytes in organ culture: Analysis of CD4/CD8‐defined single‐positive and double‐negative cells. J. Immunol. 141:355‐362.
  Describes kinetic analysis of thymic organ culture for phenotype and cell recovery and phenotypic analyses, including multiparameter flow cytometric profiles of thymocytes for a number of cell‐surface antigens.
   Jenkinson et al., 1982. See above.
  Initial, detailed description of the effects of 2‐dG treatment.
   Watanabe et al., 1989. See above.
  Comparison of seeding thymic rudiments us ing hanging drop versus microinjector techniques (measurements of cell input required, cell recovery, and phenotype).
   Ueno et al., 2005. See above.
  Describes the methods of fetal thymus organ culture in detail, including additional techniques of time‐lapse visualization and retrovirus‐mediated gene transfer.
   Zúñiga‐Pflücker, J.C. 2004. T‐cell development made simple. Nat. Rev. Immunol. 4:67‐72.
  Provides a comprehensive perspective on the OP9‐DL1 system.
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