FTOC Protocol 小鼠胚胎胸腺培养

ProtocolFetal Thymus Organ CultureINTRODUCTIONThe generation of functionally competent T-cells from their progenitors involves a series of developmental events including proliferation, differentiation, and survival. T-cell development is a non-cell-autonomous event, and requires interactions with thymic stromal cells. Fetal thymus organ cultures provide an in vitro system in which isolated embryonic thymus lobes can be maintained in culture, allowing the study of T-cell development as well as thymic stromal cell function. This system remains the only in vitro system that supports a complete program of T-cell development, including positive and negative selection of the developing T-cell receptor repertoire. Modifications of the basic fetal thymus organ culture system, such as hanging drop cultures and reaggregate thymus organ cultures, provide useful systems to analyze thymus colonization and thymic stromal cell function, respectively.MATERIALSReagentsAll media should be prewarmed to 37°C before use.10% CO2 in air, contained in gas cylinder2'-deoxyguanosine (dGuo) (Sigma-Aldrich, D0901)Prepare a 9 mM stock in 1X PBS. It takes ~1 h at 37ºC for the dGuo to dissolve.Mix the solution well during this period.Dilute the dGuo stock to a final concentration of 1.35 mM in1X PBS (see Step 3).Dulbecco’s modified Eagle’s medium (DMEM) for thymus organ culture70% ethanolMice, pregnant female (gestational age E14-E16)1X PBS (Ca++/Mg++-free)RF10-H medium10X trypsin (Sigma-Aldrich,T4674)Prepare a 1X trypsin solution by diluting the 10X stock in Ca++/Mg++-free PBS containing 0.02% EDTA.EquipmentArtiwrap sponges, 1 cm2 (Medipost Ltd.)Aspirator tube assembly (Sigma-Aldrich, A5177)Boxes (rectangular, plastic) with fitted lids (Watkins and Doncaster,E6052)Bunsen burner, fish-tailFilters (isopore membrane) with 0.8-µm pore size (Millipore,ATTP01300)Forceps, watchmaker, Dumont #5 (TAAB)Incubator, preset to 37°CMicrocentrifugeMicrocentrifuge tubes, 1.5 mLMicropipette, 1 mLMicroscope (stereo-dissecting) with magnification range 0.8X-5X(e.g., Zeiss, Stemi SV)Petri dishes, 90-mm diameter, sterile bacteriological grade(Sterilin)Plate inserts for multiwell plates (e.g., for a six-well plate,use Millicell 0.4-µm plate inserts; Millipore, PICM03050)(optional; see Step 2.i)Plates, Terasaki (Sterilin)Scissors, surgicalTapeTubing, glass, to make capillary pipettes (Fisher Scientific UK, FB51460)Vortex mixerMETHODThe standard fetal thymus organ culture (FTOC) method described in Steps 1 and 2 can be modified to study thymus colonization using hanging drop cultures (Steps 3-5; Jenkinson et al. 1982).In addition, reaggregate thymus organ cultures (RTOC) can be used, in which three-dimensional organ cultures are generated from defined mixtures of thymic stromal cells and thymocytes(Steps 6-16). This latter method is particularly useful in studying thymic stromal cell function and the development of an individual cohort of thymocytes at a defined developmental stage.Fetal Thymus Organ Culture (FTOC)1. To dissect fetal thymus lobes:i. Swab the abdominal region of a sacrificed female mouse using70% ethanol.Reflect the skin using scissors and forceps, and cut through the abdominalwall to reveal the uterus. Separate the uterus from its attachments at the two uterine horns laterally,and from the bladder anteriorly.ii. Transfer the uterus to a 90-mm Petri dish. Using scissors and forceps, cut open the uterus lengthwise to remove the embryos.iii. Place the embryos in a Petri dish containing 1X PBS to wash off any blood.Use forceps to remove the placenta and membranes from each embryo.iv. Place the embryos in a Petri dish containing RF10-H medium.Decapitate the embryos by pinching them with forceps just below the chin.Place each embryo on its back,and open the anterior surface of the chest wall by placing the tips of a closed pair of forceps into the chest cavity and allowing the forceps to open.v. Under the dissecting microscope,remove the entire thoracic tree--heart, lungs, trachea, thymus lobes--by grasping below the heart.Place the thoracic trees in a Petri dish containing fresh RF10-H medium.Problem 1: When removing the thoracic tree from the embryos, thymuslobes are left in the chest cavity.[Step 1.v]Solution:It is important to grasp the heart (a red, apple-shaped organ) firmly. Sharp straight forceps also help; if forceps are blunt or bent, fine dissection is difficult.vi. Check each tree for the presence of the two thymus lobes.Remove the thymus lobes from the thoracic tree using forceps.Thymus lobes appear as oval encapsulated organs on either side of the trachea (Fig. 1 ).Problem2:Instruments become sticky with congealed blood and tissue during the preparation of large numbers of embryos, making it difficult to manipulate dissected lobes.[Step 1.vi] Solution:Wash the instruments regularly with 70% alcohol and allow them to air-dry before use.i. Place 4 mL of prewarmed DMEM in a 90-mm Petri dish, and use forceps to place two 1-cm2 Artiwrap sponges and two 0.8-µm filters into the dish.Make sure that the medium wets the filters from below.See Troubleshooting. As an alternative to the sponge-and-filter method, multiwell plate inserts with membranes of the appropriate pore size can be used.Problem3: Large areas of the bottom of the Petri dish are not covered by medium.[Step 2.i]Solution: Make sure the volume of medium is accurate; insufficient medium in the dishes can adversely affect organ cultures. This may also be due to sponge supports that are too large and consequently soak up the medium.Problem4: Filters slip off the sponge supports and sink.[Step 2.i]Solution: Adding too much medium to the Petri dish increases the depth of the medium, which can cause filters to float off their support and sink. Make sure the volume of medium is accurate.Also, use sponge supports that are larger in area than the filters.ii. Prepare a finely drawn glass pipette by heating the glass tubing in a Bunsen flame, and allow the pipette to cool before attaching to the aspirator tube.ing a mouth-controlled glass pipette from Step 2.ii,pick up the thymus lobes individually and transfer them to the surface of the filters.See Troubleshooting. As an alternative approach to the glass pipette method, fine forceps can be used.Problem5: A large volume of medium is transferred to the filter during the placement of lobes on the filter.[Step 2.iii]Solution:Use a fine mouth-controlled glass pipette to transfer the lobes. The diameter of the glass pipette should be approximately half the size of the thymus lobe. If forceps are used, medium is easily transferred to the filter, which can submerge the lobes.iv.Place up to three 90-mm Petri dishes into a plastic rectangular box containing H2O and a Petri dish lid as a support. Gas the boxes for 10 min witha mixture of 10%CO2 in air, seal with tape, and incubate at 37ºC.The length of incubation depends on the experiment; it is typically5-10 d.See Troubleshooting.Hanging Drop Cultures3. Isolate and set up in organ culture E14-E15 fetal thymus lobes asdescribed in Steps 1 and 2. To eliminate endogenous thymocytes from the lobes, grow organ cultures in the presence of 1.35 mM 2'-deoxyguanosine (dGuo) at 37°C for 5-7 d (Jenkinson et al. 1982).4. Resuspend the thymocyte population of interest (e.g., fetal liver T-cellprogenitors, CD4–8– thymocyte subsets)in 20 µL of prewarmed DMEM, and transfer the cell(s) to a single Terasaki well. The number of the transferred cells at the outset of culture can be as few as one per well, but is typically around 2000. Place a single dGuo-treated lobe （？）in each well, and invert the plate by turning it over, so that the medium forms a hanging drop in the plate.5. Culture overnight,as for FTOC (see Step 2.iv). Colonized lobes can then beremoved from the Terasaki plate and analyzed immediately to study thymus colonization, or transferred to normal FTOC to reveal the developmental potential of the introduced cells.Reaggregate Thymus Organ Cultures (RTOC)6. Prepare dGuo-treated FTOC (see Step 3), and transfer the lobes to a dishof prewarmed RF10-H medium.7. Using a 1-mL micropipette, transfer the lobes to a 1.5-mL microcentrifugetube with no more than 20 lobes per tube, and allow them to sink to the bottom.8. Remove the medium, and wash the lobes three times in 1-mL volumes ofprewarmed 1X PBS.9. Resuspend the lobes in 600 µL of prewarmed 1X trypsin solution,andincubate the lobes at 37ºC for 10 min.10. Pipette the lobes using a 1-mL micropipette, and return them to theincubator for an additional 5 min.11. Pipette the lobes again until they completely disperse.Neutralize thetrypsin by adding400 µL of RF10-H medium.Centrifuge and resuspend the cells in 1 mL of RF-10H medium.Count the cells.Problem6: dGuo-treated thymus lobes do not digest completely in trypsin.[Step 11]Solution:It is important to digest the lobes in small batches (typically 20 lobes) rather than as one large batch. If they are not digested completely after 15 min, transfer the lobes back to 37ºC for an additional 5 min.12. Mix together appropriate numbers of thymocytes and stromal cells in a1.5-mL microcentrifuge tube, and centrifuge.To prepare RTOC, thymic stromal cells obtained as above need to be mixed with the desired thymocyte population (Jenkinson et al. 1992).In a typical experiment, ~7.5 x 105 thymic stromal cells can be mixed with 7.5 x 105 CD4+8+thymocytes.13. Remove the supernatant from the cell pellet.It is essential that all of the liquid is removed from the cell pellet prior to reaggregation.14. Vortex the cell pellet for 5 sec.This disrupts the pellet and forms a thick cell slurry.15. Using a mouth-controlled glass pipette (from Step 2.ii),transfer theslurry to the center of a 0.8-µm filter in a Petri dish.Where mouth pipetting is not possible, the cell slurry can be drawn up into a small pipette tip using a hand-controlled micropipette,and gently expelled onto the filter surface. See Troubleshooting.Problem7:When making RTOC, the cell slurry spreads out on the filter surface and intact lobes fail to form.[Step 15]Solution: If even a small volume (2 µL) of liquid is left on the cell pellet prior to reaggregation, the slurry is too thin and will spread out. During Step 13, remove the supernatant in stages using a micropipette set at 200 µL.16.Culture as for standard FTOC (see Step 2.iv).DISCUSSIONThe use of isolated thymus lobes between E14 and E16 is optimal for fetalthymus organ culture. Thymus lobes after E16 of gestation are large, a factor that often can lead to considerable necrosis in the explanted tissue.Maintaining the organ cultures in a4-mL volume of culture medium allows thecultures to be kept for several weeks without the need for replacement of the spent medium. While cultures can be maintained in CO2 incubators,the use of sealed tissue culture boxes that are individually gassed results in cultures that are unaffected by repeated openings of incubator doors. Humidity is also maintained, which helps prevent evaporation of the medium in longer-term cultures; thus,fetal thymus organ cultures can be maintained in these culture conditions for several weeks. As a general rule, if E15 fetal lobes are explanted, maturation from the CD4–8–to the CD4+8+ stage is readily detectable after 4 d in culture.Cohorts of CD4+8– and CD4–8+ cells first appear around days 7-8 of culture, and can be used to study selection events operating on CD4+8+ thymocytes. For reaggregate thymus organ culture, the number of dGuo FTOC lobes used depends upon the experiment being performed. Typically, in order to study the positive selection of a cohort of CD4+8+ thymocytes, we would use 10-15 E15 dGuo FTOCs to generate a singe RTOC, and harvest after 5 d of culture, when cohorts of CD4+8– and CD4–8+ cells are detectable.Through the use of mouse fetal thymus organ culture (FTOC) techniques, Robinson and Owen (9) showed that functionally competent T cells could be generated within explanted thymic tissue under controlled conditions in vitro, paving the way for the study of mechanisms regulating T cell development.By adopting approaches that had previously been described and used to study embryonic organ development, including thymus (10–12), Robinson and Owen described a method in which embryonic mice at embryonic days 14 and 15 of gestation were used as a source of thymus lobes, which at this developmental stage contained only large blast-like cells thought to represent lymphocyte precursors. After explantation into organ culture on the surface of floating membrane filters, thymic cultures were shown to undergo significant increases in size, correlating with an increase in lymphocyte recovery of up to 14-fold by day 7 of culture. Such culture conditions were also shown to support the emergence of lymphocytes with a size typical of peripheral lymphocytes, as well as the acquisition of the surface marker Thy-1. Importantly, B cells were found to beabsent from thymus organ cultures, an important indication that the specialization of the thymus for the production of T cells in vivo could also be recapitulated in vitro. Perhaps most significantly, Robinson and Owen also treated lymphocytes generated in FTOC with the T cell mitogens PHA and Con A, together with 125 I-labeled iododeoxyuridine. This experiment was important for two reasons. Firstly, it showed that function-ally competent T cells capable of undergoing proliferation in response to stimulation could be generated in vitro in the context of the thymic microenvironment. Secondly, it also showed the ease with which the FTOCs and the T cells generated within them could be manipulated by the addition of exogenous reagents that could enable the study of stages in Tcell development. For example, the authors went on to show that FTOC also generated T cells that responded to alloantigens in mixed leukocyte culture (13) and that tolerance to alloantigens could be induced by co culturing thymus with fragments of histoincompatible spleen (14).Since its initial description, the FTOC system has been used extensively to study the mechanisms regulating key events that occur during intra thymic T cell development. For example, FTOC proved to be a key system in unraveling the role of MHC-bound peptides in the positive and negative selection of the developing TCR repertoire by enabling study of the effects of individual peptides on a relatively synchronous wave of thymocyte development in the absence of effects on peripheral T cells (15–16). In addition, the “standard operating procedure”for FTOC has undergone several revisions that have allowed study of the role of the thymic microenvironment in T cell production, including thymic epithelial cell development and function. The ability to generate a lymphoid FTOCs by exposure to 2-deoxyguanosine(17) and the production of reaggregate thymus organ cultures from defined stromal components (18) are notable in this context, the latter currently being used as a model to study thymocyte migration and development in real time using two-photon microscopy (19).In summary, the description by Robinson and Owen (9) of an in vitro system to study T cell development has proved to be a cornerstone for our understanding of thymic function and the stages and events that occur during T cell development. That this system still remains the only in vitro system capable of supporting a full program of T cell development, from progenitor recruitment to MHC restriction and clonal deletion is a testament to the use of simple experimental approaches to study complex biological events.。