Irma Thesleff and Carin Sahlberg 1. Introduction
Interactions between epithelial and mesenchymal tissues constitute a central mechanism regulating the development of most embryonic organs. Studies on the nature of such interactions require the separation of the interacting tissues from each other and the follow-up of their advancing development in various types of recombined explants. The tissues can be either transplanted and their development followed in vivo, or they can be cultured as explants in vitro. Although the transplantation methods offer certain advantages, including physiological environment and the possibility for long-term follow-up, organ culture techniques are superior in many other aspects. The cultured tissues can be manipulated in multiple ways, and their development can be continuously monitored. The culture conditions are reproducible, and the composition of the medium is known exactly and it can be modified. Furthermore, the in vitro culture conditions allow analyses of the nature of the inductive signals.
Many types of organ culture systems have been used over the years for studies on embryonic organ development. The Trowell method (1) has been widely applied, and it has proven to be suitable for the analysis of the morphogenesis of many different organs (2-6). In this system, the explants are cultured in vitro at the medium/gas interface on thin membrane filters that are supported by a metal grid. We have used the Trowell technique as modified by Saxen (7).
The embryonic tooth is a typical example of an organ in which reciprocal epithelial-mesenchymal interactions regulate morphogenesis and cell differentiation (8). We have used the Trowell-type organ culture method for the analysis of the mechanisms of tissue interactions at various stages of tooth development (9-11). In the following, we describe the protocols for separation
From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ
and culture of dental epithelial and mesenchymal tissues, and for bead experiments in which the local effects of growth factors and other diffusible molecules can be analyzed (Fig. 1) (12-15).
All solutions and equipment should be sterile. The glassware and metal instruments should be autoclaved and solutions filtered or autoclaved. All work should be done in a laminar flow hood; also the dissection microscope should be placed in hood.
2.1. Salt Solutions, Enzymes, and Culture Media
1. Phosphate-buffered saline (PBS), pH 7.4: Dulbecco's phosphate-buffered saline (D-PBS, Gibco-BRL Laboratories, Detroit, MI, cat. no. 14080-048) supplemented with penicillin-streptomycin (PS), 20 IU/mL.
2. PS (Gibco-BRL, Life Technologies, Paisely, UK, cat. no. 15148-186), 10,000 IU/mL.
3. Enzyme solution for tissue separation: trypsin (Difco, cat. no. 0152-15); stock solution of pancreatin (Gibco BRL, cat. no. 15725-013), 1.25 g/mL; Tyrode's solution: NaCl (8.0 g), KCl (0.2 g), NaH2PO4 (0.05 g), glucose (1.0 g), NaHCO3 (1.0 g). Adjust pH to 7.2, make up to 1000 ml with distilled H2O, and sterile filter. Store at +4°C. Dissolve 0.225 g trypsin in 6 mL Tyrode's solution on ice, using a magnetic stirrer. Add 1 mL pancreatin and 20 ||L PS. Adjust pH to 7.4 with NaOH. Make up to the final volume 10 mL with Tyrode's solution and sterile filter. Aliquot 1 mL in Eppendorf tubes and store at -20°C. The enzyme solution can be stored at -20°C for 1 wk.
4 Medium for tissue dissection and culture: Dulbecco's Modified Eagle Medium with glutaMAX-1 (DMEM, Gibco-BRL, cat. no. 61965-026), supplemented with 10% heat-inactivated fetal calf serum (FCS) and PS (20 IU/ml). Store at +4°C (see Note 6).
1. For tissue dissection: 10-cm diameter plastic bacteriological Petri dishes and 410 cm diameter glass Petri dishes, small scissors and forceps, disposable 20- and 26-gage needles attached to 1-mL plastic syringes (see Note 1).
2. Culture dishes: 3.5-cm diameter plastic Petri dishes (bacteriological or cell culture dishes).
3. Metal grids: Prepared from stainless-steel mesh (corrosion-resistant, size of mesh 0.7 mm) by cutting approx 3-cm diameter disks and bending the edges on a cutting form to give a 3-mm height (the height of the metal grids can be altered to allow the use of more or less culture media). Holes in the grid are produced either by nails in the cutting form or by punching. The holes facilitate the examination and photography of the explants (and they are handy when transfilter cultures are prepared) (Note 5) (Fig. 2). There are commercially available organ culture dishes featuring a central well in which a metal grid (even without bended edges) can be placed (Falcon 3037, Becton Dickinson Ltd., Oxford, UK).
4. Filters: Nuclepore polycarbonate filters (Nuclepore, Pleasanton, CA). The pore size routinely used is 0.1 |im (see Note 4). The filters are cut in halves, washed in detergent, rinsed under running water for 2 h and 10x in distilled water, and stored in 70% ethanol.
5. Glass Pasteur pipets are used for transferring tissues. They are siliconized (to prevent sticking of tissues), stuffed with cotton wool, and autoclaved. Before use, they are drawn by heating to adjust to the size of the tissues. Ideally, the diameter should be the minimal to allow free passage of the tissue.
6. Beads for growth factors: Affi-Gel Blue agarose beads (Bio-Rad Laboratories, Hercules, CA) or heparin-coated acrylic beads (Sigma, St. Louis, MO) are divided into aliquots and stored at +4°C.
3.1. Treatment of Beads
Pipet agarose beads or heparin-coated acrylic beads to PBS in a Petri dish. Count
100-200 beads under the microscope, and transfer to Eppendorf tube. Spin down the
beads, and remove PBS. Add growth factors in a small volume (10-50 |L) of 0.1% bovine serum albumin (BSA) in PBS. (In general, high concentrations of proteins are used. We use FGF-4 at 20 ng/|iL and TGFp-1 at 1 ng/|iL). An equal amount of 0.1% BSA in PBS is pipetted to control beads. Incubate for 30 min at +37°C and store at +4°C. The beads can be used at least for 14 d (depending on the stability of the protein).
1. Take a sheet of Nuclepore filter from ethanol and rinse in PBS in a plastic 10-cm diameter Petri dish. Cut the filter, using small scissors and watchmaker forceps, in approx 3 x 3 mm pieces, and leave in PBS.
2. Place metal grids in 3.5-cm plastic culture dishes. Add approx 2 mL culture medium (D-MEM +10% FCS, see Note 6) by pipeting through the grid. The surface of the medium should contact the plane of the grid, but not cover it (excess medium results in floating of the filters and tissues). No air bubbles should remain under the grid (if present, they can be sucked empty with a thin Pasteur pipet). Using forceps, transfer the Nuclepore filter pieces on the grids placing them over the holes.
1. Place the mouse uterus (E 12) in a 10-cm plastic Petri dish containing D-PBS, and cut open the uterine wall using small scissors and forceps. Continue the work under the stereomicroscope with transmitted light. Remove the embryos from fetal membranes, and transfer them to a fresh dish of D-PBS. Cut off the heads using disposable needles as "knives." The needles are used during all subsequent steps of dissection. Transfer the heads to a glass Petri dish containing D-PBS, and dissect out the lower jaw. Dissect out the tooth germs of the first mandibular molar with some surrounding tissue left in place (see Notes 1 and 2).
2. With a drawn Pasteur pipet (preferably mouth-controlled), transfer the tooth germs to an Eppendorf tube. Remove most of the liquid. Melt an aliquot of pan-creatin/trypsin, and spin immediately for 30 s at 8000g. Add cold supernatant on the tooth germs and incubate on ice for 2 min. Remove most of the liquid, add culture medium, mix, and transfer the tooth germs to a glass Petri dish containing culture medium. Leave the tissues for 30 min at room temperature.
3. Gently separate the epithelia from the mesenchymes using needles and remove excess surrounding nondental tissue (see Note 3). Transfer the tissues on the Nuclepore filters in culture dishes that have been prepared in advance. Avoid air bubbles in the pipet, and avoid sucking the tissue beyond the capillary part of the pipet. Ideally, the tissues should be placed directly in their final position, but if needed, they can be gently pushed with needles.
4. Wash the beads quickly in culture medium in a glass Petri dish (they tend to stick on plastic dishes). Under the microscope, transfer the beads one at a time to the tissues.
3.4. Culture and Fixation
1. Culture the tissues in a standard incubator at 37°C, in an atmosphere of 5% CO2 in air and 100% humidity for 24 h.
2. Photograph the explants before fixation (the translucent zone cannot be seen after fixation). To avoid detachment of tissues from the filters, prefix the explants in ice-cold methanol on the grids as follows: Remove the culture medium by sucking, and pipet methanol gently on the tissues. Leave for 5 min, and transfer filters by watchmaker forceps to Eppendorf tubes for subsequent treatments (see Notes 8 and 9). Typical explants are shown in Fig. 3.
1. For tissue dissection, the disposable needles are superior to other instruments, such as scalpels or iris knives, because they need no sharpening or sterilization. The size of the needles can be chosen according to the sizes of tissues. The syringes need not be absolutely sterile and can be used many times. For best preservation of tissue vitality, dissecting should be done by determined cuts avoiding tearing. Glass Petri dishes are preferable to plastic ones during dissection of the tissues, because cutting with the needles tends to scrape the bottom of the plastic dish and loosen pieces of it.
2. The preparation and dissection of tissues should be done as quickly as possible to promote survival of the tissues. One uterus at a time should be prepared and the rest stored in D-PBS at +4°C. The dissected tissues should not be stored for long times (2-3 h max) before transfer to the culture dishes and incubator.
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