Genetic Modification and Recombination of Salivary Gland Organ Cultures

Podsumowanie

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Streszczenie

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity^1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood ⁴. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo⁵. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-111^6,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands⁸. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

Protokół

The protocol contains four major steps, as depicted in Figure 1. All steps are described in full detail. Adenovirus construction and viral purification should be performed in advance of the organ harvesting for use in the genetic transduction of dissected epithelial rudiments. All standard BSL-2 safety precautions should be followed when working with adenoviruses.

1. Mouse Embryonic Submandibular Gland (SMG) Harvesting and Microdissection

1. Euthanize timed-pregnant female mice (outbred strain CD-1 or ICR) using CO₂ narcosis, followed by cervical dislocation and harvest strings of mouse embryos at embryonic day 13 (E13, 3-5 epithelial buds) following procedures approved by the institutional IACUC committee. The day of vaginal plug discovery is designated as E0. Salivary glands can also be harvested and cultured at the E12 stage when there is a single primary bud and stalk with clefts just starting to initiate. SMGs prior to this stage require different culture conditions than those indicated here.
2. Transfer embryo strings into sterile 10 cm tissue culture dishes filled with 25 ml of DMEM/Ham's F12 Medium (F12) lacking phenol red (Life Technologies) and supplemented with 100 U/ml penicillin, and 100 μg/ml streptomycin (pen-strep, Life Technologies).
3. Using a sterile scalpel (#11 blades) and forceps (#5, Fine Science Tools, 11252-20), remove the embryos from the sacs into a separate 10 cm dish containing 25 ml of DMEM/F12 + pen-strep.
4. Sever the embryo heads with an angled cut just below the lower jaw.
5. Isolate the lower mandibles from the heads under a stereo dissecting microscope with a transmitted light base (Nikon SMZ645 or equivalent) using a cut below the upper jaw. Place the tissues on top of an extra piece of glass rather than directly on the glass component of the microscope base.
6. Collect the lower mandibles into 3 ml of DMEM/F12 + pen-strep in a sterile 35 mm tissue culture dish.
7. Place a mandible slice on the dissecting microscope stage so that the tongue is on the bottom of the slice but is pointing towards the 12:00 o'clock position.
8. Using one side of two crossed forceps in a scissoring motion, remove and discard the lower 1/3^rd of the mandible slice.
9. Turn the slice 90 ° to the right (if right-handed) and, using a scissoring cut with the forceps, cut the top portion of the mandible slice (without cutting the tongue) halfway into the slice.
10. Peel the excess tissue away and remove it to expose the SMGs underneath. There will be one on each side near the base of the tongue.
11. Using one pair of forceps to hold down the tissue by the tongue, use the other forceps to tease the SMG away from the tongue. Repeat for the other gland.
12. Collect the microdissected salivary glands into 3 ml of DMEM/F12 + pen-strep into a new sterile 35 mm tissue culture dish. Note: the larger submandibular glands (3-5 epithelial buds) along with the connected smaller sublingual gland (1 bud) can be cultured intact by skipping to step 4 and performing steps 4.1, 4.3, and 4.4, as described previously ^2,8.

2. SMG Epithelial Rudiment Separation

1. Place 5 (or more) salivary glands into a glass-bottomed, 50 mm diameter microwell dish (MatTek Corporation, P50G-1.5-14-F) containing 200 μl of Hank's balanced salt solution (HBSS lacking Ca⁺⁺or Mg⁺⁺, Life Technologies) containing 0.4% (v/v) dispase (Life Technologies, cat. no. 17105-041) and incubate for 15 min at 37 °C.
2. After the incubation, using a sterile 200 μl pipette tip, carefully aspirate out the dispase solution without disturbing the glands. Immediately add 200 μl of DMEM/F12 media containing 5% (w/v) BSA (DMEM/F12-BSA, pre-sterilized with a 0.22 μm filter) to neutralize the dispase.
3. Under a dissecting microscope, using a pair of forceps with fine tips (#5 Dumostar, Fine Science Tools, 11295-20), gently separate the loosened mesenchyme from around the SMG epithelial buds and ducts, taking care to leave the epithelium intact.
4. Pre-wet a sterile 200 μl pipette tip with DMEM/F12-BSA media and gently pipette out the epithelial rudiments into a new 50 mm diameter microwell dish containing 200 μl of DMEM/F12 + pen-strep. Use a high quality 200 μl pipette tip with a smooth edge.
5. In the dish that contains the mesenchyme pieces, discard any non-mesenchymal tissue including the sublingual glands and any detached submandibular gland buds.

3. Adenoviral Infection of Epithelial Rudiments

1. Make 200 μl of viral infection media by diluting the adenovirus of interest in DMEM/F12 + pen-strep in a microcentrifuge tube so that the titer of the resulting solution is 1x10⁸-10⁹ PFUs. The optimal titer required for different viruses may vary. It is important to use cesium chloride (CsCl)-purified viral preparations for optimal uptake efficiency.
2. Remove the media from the dish containing the five epithelial rudiments. Quickly add 200 μl of the infection media, keeping the rudiments wet at all times. Take necessary precautions when handling the virus and disposing of contaminated pipet tips. Disinfect any virus-contaminated surfaces with 10% bleach solution.
3. Move the epithelial rudiments far away from each other with forceps to prevent them from sticking together and allow them to settle to the bottom of the plate. Incubate rudiments in viral media for 1 hr at room temperature. During the incubation, separate the rudiments, if they move close together. Excessive rocking or movement will allow the glands to clump together.
4. After incubation, gently remove the viral-containing media and discard appropriately, taking care not to disrupt the rudiments. Add 200 μl of DMEM/F12 + pen-strep. Repeat this wash at least two more times and replace with 200 μl of DMEM/F12 + pen-strep media. These washes are critical to remove any virus that is not strongly attached to the epithelium and TO prevent infection of the mesenchyme.
5. Incubate epithelial rudiments with 200 ml of DMEM/F12 containing 2% (v/v) Matrigel (BD Biosciences, 356231) for 20 min. We find that this short incubation in Matrigel minimizes the regression of existing clefts during the initial culture period, but this step can be dispensed with if exposure to Matrigel is not desired.

4. Ex vivo Culture of Recombined SMGs

1. Place each rudiment, one at a time, using a pre-wet 200 μl pipette tip, on top of a notched (small cut) 13 mm, 0.1 μm Nuclepore Track-Etch membrane filter (Whatman) floating over 200 μl of culture media (5 rudiments /filter) in a glass-bottomed microwell plate. The culture media is: DMEM/F12 + pen-strep containing 50 μg/ml transferrin and 150 μg/ml ascorbic acid, as previously described^2,8. Transferrin is known to stimulate survival and branching morphogenesis of salivary gland and other organ explants⁹. Addition of ascorbic acid has been shown to stimulate SMG branching.¹⁰ It is known to act as a cofactor in many hydroxylation reactions of metabolic pathways (such as proline hydroxylation during collagen fiber formation¹¹) and also stimulates fibronectin and laminin production in other organ explants¹².
2. Remove as much media on top of the filter as possible after adding each epithelial rudiment. Too much media on top of the filter may cause the filter to sink, while less media makes placement of the rudiments and mesenchyme much easier. Excess media can be removed either with a pipette tip or by using forceps to grip the media between the tips.
3. Place mesenchyme pieces derived from three to four glands on top and/or around each epithelial rudiment. Remove any extra media that is surrounding the glands to avoid movement of the mesenchyme away from the rudiments. Mesenchyme derived from more glands is not beneficial but less can be detrimental to epithelial rudiment growth.
4. Incubate recombined SMGs in a humidified incubator (95% air/5% CO₂) at 37 °C for 48 -72 hr, as desired.
5. Acquire brightfield and/or fluorescent images of live glands at 0 hr (if desired), 2 hr after recombination and every 24 hr thereafter on an inverted, upright, or stereo light microscope fitted with a digital camera using a 4X or 5X magnification objective to capture the entire recombined gland in a single frame of view. Images of the salivary gland show the best contrast using either the brightfield setting or the appropriate phase ring placed part way into the light path (which is not possible on fully-automated microscopes). Standard phase contrast is not necessary since the gland is thick and creates contrast.
6. At the desired time-points, recombined SMGs can be fixed for 20 minutes with fixative solution made of 2% paraformaldehyde (PFA) in 1X PBS containing 5% (w/v) sucrose (to better preserve internal cellular structure by keeping the cells in an isosmotic state). Unlike 4% PFA, 2% PFA will preserve a significant amount of the GFP signal if the fixative is completely removed after fixation. Glands can be stored for up to 1 week in 1X PBS in the dark at 4 °C until whole mount immunocytochemistry and confocal imaging is performed, as reported previously^3,6,13-15. Ideally, immunocytochemistry and imaging should be performed soon after fixation to obtain the best quality images. Glands may also be lysed in RIPA buffer for SDS-PAGE followed by Western blotting analysis.

Wyniki

The flow of the major experimental steps is outlined in Figure 1. An example of an intact SMG, an isolated epithelial rudiment, and its corresponding mesenchyme are shown in Figure 2. Brightfield images of recombined SMGs, which continue to undergo branching morphogenesis when cultured ex vivo for the indicated times, are shown in Figure 3. Recombined glands grown for 48 hr expressing epithelial GFP are shown in Figure 4. Confocal images of reco...

Dyskusje

The ex vivo epithelial-mesenchymal recombination technique was first published for submandibular salivary glands in 1981¹⁶. In this protocol, we expand upon the original method, using adenoviral infection to manipulate epithelial cell gene expression within the context of a recombined gland. We show that a percentage of the epithelial cells are infected with the adenovirus, whereas the percentage of cells that are infected depends upon the properties of the viral promoter, viral titer, and vira...

Materiały

Name	Company	Catalog Number	Comments
Name of the Reagent	Company	Catalog Number	Comments
DMEM/Ham's F12 Medium without phenol red	Life Technologies	21041-025
Penicillin and Streptomycin	Life Technologies	15070-163	10X stock
Dispase	Life Technologies	17105-041	Freeze single use aliquots at -20C
BSA	Sigma	A2934-100G	Fraction V, low endotoxin
Adeno-X-GFP	BD Biosciences	8138-1	Should be high titer (1x1010 pfu/ml). CsCl purified viruses are more effective than column-purified viruses in this assay.
16% Paraformaldehyde	Electron Microscopy Sciences	15710	Diluted to 2% in PBS with 5% sucrose (w/v)
1X Phosphate-buffered saline (PBS)	Life Technologies	70011-044	Prepared from 10X stock
Hank's Balanced Salt Solution	Life Technologies	14175095	no Calcium, no Magnesium, no Phenol Red
Transferrin	Sigma	T8158	25 mg/ml stock solution in DMEM/F12 media. Freeze single-use aliquots at -20C
L- Ascorbic acid (Vitamin C)	Sigma	A4403	75 mg/ml stock solution in DMEM/F12 media.Freeze single-use aliquots at -20C
			Table 1. List of reagents required for SMG recombination protocol.
10 cm sterile plastic dishes	Corning	430167	Non-tissue culture-treated plates can also be used.
Stereo dissecting microscope with transmitted light base	Nikon	SMZ645	Any stereo dissecting microscope can be used that has a transmitted light base.
35 mm tissue culture dishes	Falcon	353001	Non-tissue culture-treated plates can also be used.
50 mm diameter microwell dishes	MatTek Corporation	P50-G-1.5-14F
Nuclepore Track-Etch membrane filters	Whatman	110405	13 mm diameter, 0.1 mm pore size
Widefield fluorescence microscope	Carl Zeiss, USA	Axio Observer Z1	Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached digital camera.
Confocal microscope	Leica Microsystems	TCS SP5	Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.
Timed-pregnant female mice, strain CD-1 or ICR	Charles River Labs		Embryos are harvested on day 13 (with day of plug discovery designated as day 0).
Scalpel blade #11	Fine Science Tools	10011-00
Scalpel handle #3	Fine Science Tools	10003-12
Dumont #5 forceps inox alloy, 0.05mm X 0.02mm	Fine Science Tools	11252-20	Ideal for harvesting glands from embryos
Dumont #5 forceps dumostar alloy, 0.05mm X 0.01mm	Fine Science Tools	11295-20	Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used.
			Table 2. Equipment used in SMG recombination protocol.