Direct tissue explant method for derivation and study of extravillous trophoblast

From early post-implantation stages to approximately 18 weeks' gestation, cytotrophoblast at the tips of anchoring villi at the placental periphery proliferate to form columns. From the distal edges of these, single cells break away and infiltrate the adjacent maternal decidual stroma, progressing as far as the inner one third of the myometrium. These cells invade maternal spiral arteries and transform their walls with loss of smooth muscle and the associated elastic and collagenous extracellular matrix which is replaced by a fibrinoid substance.

This method enables the researcher to explant terminal placental villi onto a three dimensional matrix of either collagen or Matrigel and monitor the formation of anchoring cytotrophoblast columns. The resulting cells can be stimulated to assume a migratory phenotype by growth factors. TGFb1 inhibits outgrowth.


Explant culture of first trimester placental tissue for de novo generation of extravillous trophoblast

The protocols described include explant on either collagen I or Matrigel. The results are similar, but trophoblast outgrowth on collagen occurs radially at the surface while on Matrigel it occurs predominantly in a downward direction into the gel. The former experiment is therefore more conveniently monitored by whole mount microscopy (Figure 6). Under control conditions of 20% oxygen and serum free medium, cell proliferation occurs during the first 24 h after which it ceases and further outgrowth on collagen, which continues for up to about 5 days, represents the reorganisation of an existing pool of cells from a thick column at the villous tip to a sheet only a few cells thick at the collagen gel surface.

Explant culture method

Equipment and reagents

• 6, 12 or 24 well culture plates (Costar)
• Pastettes
• 9 cm petri dishes
• 5 cm petri dishes
• Rat tail collagen type I (Becton Dickenson, distributed by Stratech)
• Matrigel (Becton Dickenson, distributed by Stratech)
• 10 x DMEM (Sigma)7.5% sodium bicarbonate (Gibco/BRL)
• Culture medium: 1:1 mixture of DMEM and Ham's F12
• 25 gauge needles
• 1 ml syringes
• 10 ml stripettes
• Antibiotic, antimycotic mix AAM (Sigma)
• Fetal calf serum (Gibco/BRL)
• Growth factors
• Humid box
• 70% ethanol
• 50 mg collagenase (Sigma type IA)

Method

Collagen gel

Collagen gels should be prepared the day before the experiment. Storing gels for a longer time period than may result in deterioration as indicated by a stringy appearance. For each 12 well plate, 1 ml of collagen type I is required. Ideally the collagen concentration should be more than 3 mg/ml. Gels become less firm with storage time of the collagen, and more dilute stock solutions tend to have a shorter shelf life. If more than 3-4 ml of collagen are to be used then it is recommended that it be prepared in separate 1-2 ml batches to ensure even mixing. For best results use collagen, 10x medium and sodium bicarbonate directly from the fridge.
For 1 plate:

1. Withdraw 1 ml of collagen from the container using a 1 ml syringe and needle. Discard the needle before transferring to a 1.5 ml Eppendorf tube (this prevents the introduction of air bubbles).
2. Add 100 ml of 10x DMEM and mix carefully with a disposable pastette, taking care not to introduce bubbles (solution is a yellow colour).
3. Add approximately 200 ml of 7.5 % sodium bicarbonate to neutralise the collagen and cause it to set (the colour changes from yellow to pink). Again, mix this carefully with a pastette as the solution will set from the bottom.
4. Use the pastette to transfer 1 drop (approximately 80 ml) to the centre each well of the plate. Avoid including air bubbles by careful pipetting. The culture plate can either be left in the hood or carefully transferred to a 5% CO2 incubator. The collagen should take around 10 minutes to gel.
5. Then add culture medium to each well to cover the gel and incubate the plate in humid box at 37°C in a CO2 incubator until required.

Gels can alternatively be set using ammonia. This is accomplished by making up the solution as above and spotting into the plates which are then carefully placed inside a box. A few drops of ammonia are placed on a small petri dish inside the box which is then sealed and incubated for 15-30 minutes at 37°C. Care must be taken not to disturb the collagen droplets and cause them to spread - they will be more fluid than in the normal method -- and not to let the gels dry out. After setting the gels are incubated as before in a box free from ammonia vapour.

Matrigel

Matrigel should be prepared at least 24h prior to setting up explant cultures. Matrigel sets at room temperature, therefore all preparation should be performed on ice or at 4°C.

1. Frozen (-20°C) aliquots of Matrigel should be defrosted overnight at 4°C. Pipette tips and culture plates should also be pre-cooled to 4°C to prevent premature gelling of the Matrigel.
2. Mix 1 ml of Matrigel with 100 ml of 10x DMEM in a 1.5 ml Eppendorf and start the gelling process by adding 200 ml of 7.5% sodium bicarbonate ensuring complete mixing before placing a drop of approximately 80 ml of Matrigel mix in the centre of each culture well.
3. Incubate the culture plates at 37°C for 10 -15 min and then cover the gel in 1 ml of SFM, and store in a sealed humid box at 4°C until required.
   

Selection of tissue

Using scissors, cut numerous terminal portions (~2-3 mm) of the villous tree for explant culture. For optimal results, villi should be either trimmed from the placental periphery under a dissecting microscope or the pieces checked for suitability using the microscope. Optimal attachment is obtained when villous clusters include several branches. Observe the frond like structure of the villi after gentle teasing out with fine forceps. If the mesenchymal (terminal) villi appear elongated and stringy to the naked eye, then success of attachment and outgrowth tends to be low. Use a portion that includes an intermediate villus with several mesenchymal branches emanating from it. Selected portions can be placed in a drop of culture medium in a petri dish until enough material has been collected.
Explant Attachment

1. Once the placental tissue has been dissected into appropriate sized pieces, drain the gels completely of medium; during incubation they will have contracted slightly and become firm, aiding tissue attachment.
2. Transfer one piece of villous tissue to each well using fine forceps and carefully tease out the branches over the gel surface, taking care not to score the gel. Alternatively, leave the medium in the well and add the selected villous tissue to the medium.
3. Carefully drain the medium from the well gently and lower the tissue onto the collagen drop. If correct placement does not occur then use a pastette and a minimal volume of medium to gently lift and place the villous tissue onto the drop. This method avoids potential scoring of the collagen gel. Ideally the villous termini should be arranged radially over the surface of the gel drop.
4. Following placement, cover each piece of tissue with 20 ml of pre-warmed serum- free culture medium and incubate at 37°C in 5% CO2 in a humid box for 3 -12 hours. After this period, initial outgrowth is sometimes seen using a high magnification phase contrast objective. For maximum attachment, the villi are incubated overnight at 37°C with 50 ml of serum-free medium. Attachment may be indicated by the appearance of radial stress lines of bundled collagen in the gel. Following this initial period, gently immerse cultures in 1 ml of serum-free medium, directing a pipette to the side of the culture well to avoid detachment of the tissue from the gel. At this stage growth factors, bromodeoxyuridine or adhesion-modulating antibodies may be added to the explant in the serum free medium at suitable concentrations (32).
5. Transfer the culture plate carefully back to the humid box in the incubator. After a further 12 h, initial outgrowth from viable attached terminal villi structures should be apparent. If tissue becomes detached when 1 ml medium is added or during the overnight incubation that follows, then reattachment can be attempted by draining the well and repositioning the explant. However, repositioned tissue often has poor viability and is likely to detach again as well as exhibiting delayed growth characteristics.

Cytotrophoblast Column Growth

Cytotrophoblast column outgrowth and subsequent cell migration from gel-attached villi is monitored daily using low and high power whole mount microscopy. For daily comparisons of column growth, low power image capture is recommended (Figure 6). The area of outgrowing cells is very variable, being highly dependent on the size of the initial villus-gel contact (32). Outgrowth is limited to the tips of the villi but cells from adjacent tips merge to form a shell-like structure surrounding the explant (35).

Recovery of cytotrophoblasts

1. Removal of tissue from the surface of a collagen gel after up to 7 days' culture leaves a pure population of extravillous cytotrophoblast at the surface. To release cytotrophoblasts, remove the explant conditioned medium, filter and store it at -20°C, and wash the explant 3x with PBS.
2. Remove the villous tissue from the collagen with fine forceps and incubate the remaining cells with 100 mg/ml collagenase (Sigma type 1A) at 37°C. Collagenase should be reconstituted in DEPC-treated sterile PBS if the isolated cytotrophoblasts are to be used for RNA extraction. Cytotrophoblast cells are released from the collagen by a 10min incubation at 37°C. Gentle agitation by pipetting can aid cell release.
3. Pellet the cytotrophoblasts by centrifugation at 1500 rpm for 10 min at 4°C, and wash with PBS twice to remove all traces of collagenase.
4. Trypsinisation is also an effective method for recovery of cytos.