Protocol

Preparing Recombinant Gonad Organ Cultures

Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA

¹ Corresponding author (b.capel{at}cellbio.duke.edu)

INTRODUCTION

It can be useful to assay migration between any two adjacent tissues during development. This protocol assays cell migration between the gonad and mesonephros using tissue recombination between genetically marked and unmarked tissue, combined with an organ culture technique. First, agar blocks are prepared in a custom-built mold. The size and shape of the wells are important to maintain the authentic three-dimensional morphology of the organ; the molds here are designed specifically to accommodate the gonad/mesonephros complex. Freshly dissected organs are then transferred to grooves within the agar blocks, where they are allowed to grow over 24-48 h. Using this protocol, organs develop with good morphology, and show only an ~12-h delay relative to in vivo development.

RELATED INFORMATION

Details on the techniques used in this procedure have been described previously (Martineau et al. 1997; Tilmann and Capel 1999; Brennan et al. 2002, 2003). An overview of the preparation of recombinant organ cultures is presented in Figure 1 . The sex of the embryos can be determined by Sex Chromatin Staining in Amnion Cells (Capel and Batchvarov 2008).

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Figure 1. Overview of experimental plan. The gonad/mesonephros complex is dissected from wild-type (CD1) and genetically marked mice (ROSA 26). Gonads and mesonephroi are separated, and wild-type gonads are recombined with transgenically marked mesonephroi and cultured for 24-48 h in agar blocks.

MATERIALS

Reagents

Bacto Agar

Buffer for DNA extraction

DMEM containing 10% fetal calf serum and 50 µg/mL ampicillin (organ culture medium)

Dulbecco’s Minimal Eagle’s Medium (DMEM), prewarmed to 37°C for Step 17

Embryos, mouse, 11.5-16.5 days post-coitum (dpc)

Obtain embryos from pregnant mice of both a wild-type strain and a transgenic strain expressing a constitutive marker in all of its cells (e.g., β-gal [Gt(ROSA)26Sor/J] or EGFP [Cg-Tg(CAG-EGFP)B5Nagy/J]; both lines are available from the Jackson Laboratory.)

Ethanol (optional; see Step 8)

Equipment

Agar-block molds

The dimensions of the wells are important to maintain the morphology of the organ. Design wells specifically for the organ under study, and have them custom-made in a good machine shop. For the recombinant gonad culture presented here, use the dimensions in Figure 2 .

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Figure 2. (A) Dimensions of machined mold for casting agar blocks (in.). (B) Finished mold. This mold has three ridges of slightly different dimensions.

Autoclave

Beaker

Dishes (tissue culture; 30-, 60-, and 100-mm)

Erlenmeyer flask (500-mL)

Forceps (#5; see Step 16)

Hood (tissue culture)

Incubator (tissue culture) preset to 37°C and 5% CO₂

Micropipettor with tips (20-µL, 200-µL)

Microscope (dissecting)

Needles (30-gauge)

Pipettes (glass, hand-pulled)

The diameters of the openings should be slightly larger than the mesonephroi or gonads, as appropriate; see Steps 21 and 24.

Pipettes (plastic, transfer)

Trim tips to accommodate the urogenital complex comfortably.

Plates (tissue culture; four-, 12- or 24-well)

Razor blade (sterile)

Soap for washing agar-block molds

Stir bar

Stirrer/heater

Tubes (heatproof, 15 mL)

Tubes (microcentrifuge, 1.5-mL) (for 11.5 dpc wild-type embryos; see Step 15.i)

METHOD

Preparation of the Agar

1. Add 1.5 g of Bacto Agar to 100 mL of DMEM in a 500-mL Erlenmeyer flask. Add a stir bar.

2. Place the flask into a beaker of H₂O to make a double boiler. Heat while stirring on a stirrer/heater until the agar is dissolved completely.
Do not microwave; it will overflow.

3. Transfer the flask directly to the heat. Boil the agar for 3-5 min.
Monitor carefully; this boils over easily.

4. Aliquot the agar into 15-mL heatproof tubes in convenient quantities.
Use 5-9 mL, depending on the number of blocks needed for an experiment. Each block requires 1.5 mL.

5. Store the agar aliquots at 4°C.

Preparation of the Agar Blocks

6. Wash the agar-block molds with soap and water. Rinse 10 times with tap H₂O and 10 times with distilled H₂O.

7. Autoclave the molds.
Alternatively, just before use, dip the blocks in ethanol and flame them.

8. Reheat an aliquot of agar in a beaker of H₂O until it melts completely, then remove from heat and keep in a beaker of warm H₂O.

9. Working in a tissue culture hood, place a mold with the trench side up into a 100-mm tissue culture dish.

10. Slowly pipet 1.5 mL of melted agar onto the top of the mold (surface tension will keep the agar on the block).

11. Cover the tissue culture dish to avoid dehydration. Allow the agar to set for ~10 min.

12. Using a sterile razor blade, loosen the agar around the edges. Peel the agar off of the block, inverting it into a 30-mm tissue culture dish.
Peel the agar from the block in the same direction as the trenches are running or the agar will break along the trenches.

13. Cover the agar with 1.5 mL of organ culture medium. Allow the agar to equilibrate for at least 2 h (or overnight) in the incubator.

Assembling Recombinant Gonad Organ Cultures

14. Collect embryos from wild-type or transgenic mice at the developmental stage of interest. Place each embryo in a single well of a tissue culture plate (four-, 12- or 24-well).

15. For 11.5-dpc wild-type embryos, follow these additional steps:

i. Collect the amnions into 1.5-mL microcentrifuge tubes on ice.

ii. Catalog each amnion with the well number containing the associated urogenital complex (see Step 17).

iii. Determine the sex of the embryos using the protocol Sex Chromatin Staining in Amnion Cells (Capel and Batchvarov 2008).
This is a quick method for identifying XX samples by the presence of cells with a condensed chromatin body.

iv. Reserve a bit of each embryo’s tail for confirmation of the sex by PCR.
Tail tips are stable in a dry tube on ice for several hours during dissection. Alternatively, they can be processed immediately for DNA extraction or stored at -20°C in buffer for DNA extraction.

16. Using #5 forceps, dissect the entire bilateral urogenital complex (see Fig. 3 ) along with the dorsal aorta from all of the embryos.

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Figure 3. The entire urogenital complex at 11.5 dpc contains the left and right gonad (G)/mesonephroi (M) and the dorsal aorta (DA), which lies at the midline. Separate the left and right gonad/mesonephros from the complex when you are ready to assemble to avoid tissue curling. Separate the gonad and mesonephros using a 30-gauge needle.

17. Place each complex back into the individual well of the tissue culture plate containing prewarmed DMEM. Keep tissues at 37°C and 5% CO₂.
Four-well plates are preferred: You can remove one plate at a time from the incubator for fine dissection while the rest of the samples stay at 37°C and 5% CO₂.

18. After determining the sex of the wild-type embryos, transfer each urogenital complex to a 60-mm dish using a plastic transfer pipette (trimmed to accommodate the urogenital complex).

19. Using a 30-gauge needle, separate the mesonephroi from the urogenital complexes of the embryos carrying a transgenic label.

20. Using a micropipettor with a 200-µL tip, remove all but 100-200 µL of medium from the dish holding the agar block (from Step 13).
It is much easier to assemble the recombination tissues in a dry well.

21. Using a pulled glass pipette that has a diameter slightly larger than the mesonephros, transfer the transgenic mesonephroi into the grooves of the agar blocks.
Each groove can hold approximately three mesonephroi.
Dissect one pair of mesonephroi at a time and transfer them to an agar block to prevent curling of the organ.
See Troubleshooting.

22. Position mesonephroi into the groove under a dissecting microscope (see Fig. 3).
Do not crowd samples; they are easier to work with if they are positioned far apart.

23. Using a fresh 30-gauge needle, separate the gonads from the wild-type urogenital complexes.
To prevent curling, dissect only one or two wild-type gonads before transferring them to the agar and immediately assembling with a mesonephros.

24. Using a pulled glass pipette that has a diameter slightly larger than the gonads, transfer gonads to an agar groove containing a mesonephros.
When drawing the gonads into the pipette, keep track of the "cut" side, so that you can deposit the cut side toward the cut side of the mesonephros in the well.
See Troubleshooting.

25. Assemble the cut edges of the gonad and mesonephros together (Fig. 4 ).

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Figure 4. (A) Agar block with three trenches, equilibrated, medium removed, and ready for loading samples with pulled glass mouth-pipette. (B) Four gonad/mesonephros cocultures are loaded in the top trench, and one is loaded in the middle left. (C) Higher magnification of coculture positioned in trenches; (left) gonad oriented downward; (right) gonad facing up.

See Troubleshooting.

26. Once all the samples are assembled completely, use a micropipettor with a 20-µL tip to remove the remainder of the DMEM.
Removing medium from the grooves will promote adhesion between the mesonephroi and gonads once you are satisfied with their position.

27. Add 350-400 µL of fresh organ culture medium to the bottom of the 30-mm tissue culture dish. Incubate for 1-2 h in the incubator.
Do not disturb the samples until they have had time to adhere.

28. Incubate the agar blocks in closed tissue culture dishes at 37°C in 5% CO₂.
Placing the 30-mm dishes inside 100-mm culture dishes makes them easier to handle when transferring in and out of the incubator.

29. Change the organ culture medium in the bottom of the dish daily.
Cultures maintain good morphology for at least 48 h.
See Troubleshooting.

TROUBLESHOOTING

Problem: The gonads or mesonephroi stick to the inside of the glass pipette.

[Steps 21 and 24]

Solution: Be sure to pick up a small volume of liquid in your pipette before you pick up the organ.

Problem: The gonads and mesonephroi are not properly aligned.

[Step 25]

Solution: The organs are very sticky. Consider the following:

1. Use pulled glass pipettes that are just slightly larger than the organ. This will provide better control as you pick up and orient the two tissues.

2. Make sure that the cut sides of the organs are facing one another.

3. If organs fail to align properly, add some DMEM to the trough and allow the organs to float apart. Then remove most of the medium (i.e., all but 100-200 µL) and use a pulled glass pipette or a needle to reassemble the gonads and mesonephroi.

Problem: Cultures are not growing optimally.

[Step 29]

Solution: Consider the following:

1. Cultures can be improved by pipetting a drop of medium atop each organ to wash it each time you change the medium. Remove the excess washing medium from the well before returning samples to the incubator.

2. There are differences in serum batches that affect viability and migation efficiency. When changing serum, test three or four batches to determine the best lot.

Problem: Mixed organ cultures become contaminated.

[Step 29]

Solution: Consider the following:

1. Replace the medium each day to help control contamination.

2. In general, additional antibiotics and fungicides take a toll on the tissue, but they might be required under some conditions.

3. Spread the samples among multiple plates so that all the work is not lost if one plate becomes contaminated.

Problem: Labeling of plate is lost and it is not possible to distinguish the top and bottom groove on the plate

Solution: Always load the plate asymmetrically (e.g., four samples in the top groove, two in the middle, and three on the bottom). Document the location and details of each sample in a diagram in your notebook for reference in case labels on the plates are smudged or lost.

REFERENCES

Brennan, J., Karl, J., and Capel, B. 2002. Divergent vascular mechanisms downstream of Sry establish the arterial system in the XY gonad. Dev. Biol. 244: 418–428.[Medline]

Brennan, J., Tillman, C., and Capel, B. 2003. Pdgfr- mediates testis cord organization and fetal Leydig cell development in the XY gonad. Genes Dev. 17: 800–810.[Abstract/Free Full Text]

Capel, B. and Batchvarov, J. 2008. Sex chromatin staining in amnion cells. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5079.[Abstract/Free Full Text]

Martineau, J., Nordqvist, K., Tilmann, C., Lovell-Badge, R., and Capel, B. 1997. Male-specific cell migration into the developing gonad. Curr. Biol. 7: 958–968.[Medline]

Tilmann, C. and Capel, B. 1999. Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad. Development 126: 2883–2890.[Abstract]