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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Curr Protoc Cell Biol. 2018 Nov 5;83(1):e76. doi: 10.1002/cpcb.76

Generating Embryonic Salivary Gland Organoids

Zeinab F Hosseini 1,2,#, Deirdre A Nelson 1,#, Nicholas Moskwa 1,2, Melinda Larsen 1,*
PMCID: PMC6948183  NIHMSID: NIHMS991513  PMID: 30394683

Abstract

Organoids are important research tools for studying organ morphogenesis and differentiation because they recapitulate ex vivo the native 3D organization of cells that is essential for proper cell and organ function. The composition of organoids can be manipulated to incorporate specific cell types to facilitate molecular interrogation of cell-cell interactions during organoid formation. We describe a method for generating organoids derived from both embryonic salivary gland epithelial progenitor cells and mesenchymal support cells. We describe methods for isolating enriched populations of the epithelial cells as clusters and the mesenchyme cells as single cells from mouse embryonic submandibular salivary glands. Separating the epithelial and mesenchymal cell populations allows for independent molecular manipulation of each cell type. In addition, we describe methods for lentiviral transduction of the mesenchyme cells and quantitative image analysis of organoids. The methods described here are useful for exploring mechanisms driving organ formation.

Keywords: salivary, organoid, proacinar, mesenchyme

INTRODUCTION

Organoids have emerged as major research tools for studying tissue and organ development, regeneration, and disease since they recapitulate the native 3D organization that is essential for proper cell and tissue function and because they can be manipulated to allow for interrogation of cellular and molecular mechanisms involved in organ formation. Organoids can be formed in 3D matrices from single stem or progenitor cells, from stem and progenitor cell outgrowth from tissue pieces, or from isolated stem and progenitor cell clusters (Kretzschmar & Clevers, 2016; Lancaster & Knoblich, 2014). Stem and progenitor cells can be derived from pluripotent or from tissue resident sources and many studies aim to expand these limiting cell populations for research or for therapeutic purposes. Equally important is elucidation and optimization of culture conditions to direct the differentiation of the stem and progenitor cells into functional tissues that resembles the native organ architecture that is essential for proper organ function.

In this chapter we detail methods using epithelial progenitor cells from embryonic submandibular salivary glands to produce complex salivary organoids. We describe a method for separation of the epithelial and mesenchymal populations (Basic Protocol 1) together with Support Protocol 1 to isolate more enriched epithelial clusters and Support Protocol 2 to further enrich the mesenchyme cells. In Basic Protocol 2, we describe methods to embed the epithelial clusters in basement membrane extracts to form the complex organoids that exhibit robust secretory pro-acinar differentiation with high levels of membrane-localized Aquaporin 5 (AQP5) in distal peripheral budded structures surrounding the more central ductal cell populations reminiscent of the native tissue architecture. The organoids show physiologically relevant AQP5 expression that equals or exceeds that of the starting population, prepared as described in Support Protocol 3. Importantly, inclusion of FGF2-expressing mesenchyme is critical for supporting the differentiated secretory pro-acinar cell population (Hosseini et al., 2018) in the organoids. Significantly, using highly enriched epithelial cell clusters requires adding back mesenchyme cells either distributed within a basement membrane (Alternate Protocol 1) or presented as a feeder layer (Alternate Protocol 2) to promote robust epitheial secretory pro-acinar differentiation. As manipulation of the mesenchyme cells is important for identification of signals produced by these cells, we include methods for lentiviral infection for genetic modifications of the isolated mesenchyme cells. Importantly, we also describe our methods for quantitative immunocytochemistry (ICC) of the resulting cell populations, including comparisons to the time zero input cell populations which allows comparison of protein expression and localization relative to the starting cell populations (Support Protocol 5).

STRATEGIC PLANNING

This protocol was developed for use with mouse submandibular salivary glands (SMG) isolated from the CD-1 outbred strain at embryonic day 16 (E16), with the day of plug discovery defined as day 0. The CD-1 and the equivalent outbred strain ICR, have been used extensively in studies of embryonic submandibular gland development. The protocol can be adapted for glands harvested from embryos of other ages and from other mouse strains; however, minor modifications to the protocol may be necessary. Timed pregnant females should be produced for work with embryos. Although multiple endpoint assays are possible for the organoids, we describe here a method for quantitative immunocytochemistry of the organoids as whole mounts, which we typically perform after 7 days of culture. As with all experiments involving mouse tissue, approval for the work must be first obtained by the relevant institutional IACUC committee and procedures must be performed in compliance with the approved IACUC protocol.

Lentiviral manipulation of the mesenchyme allows for interrogation of signals provided by the mesenchyme cells but adds time to the protocol. If lentiviral transduction of the mesenchyme is performed, peak expression levels are only obtained after at least 72 hrs. Co-culture of the transduced mesenchyme with fresh epithelial clusters can be performed between 3–14 days after exposure to the virus. Although third generation lentiviral vectors are relatively safe and easy to use, note that approval to work with lentivirus must be first obtained from the Institutional Biosafety Committee (IBC). Appropriate personal protective equipment must be worn and appropriate precautions must be taken when working with lentivirus.

BASIC PROTOCOL 1

ISOLATION OF ENRICHED PREPARATIONS OF EPITHELIAL PROGENITOR CELL-CONTAINING CLUSTERS SEPARATELY FROM AN ENRICHED MESENCHYMAL CELL POPULATION FROM E16 MOUSE SUBMANDIBULAR GLANDS.

To isolate SMG epithelial cells enriched for pro-acinar progenitor cells, we used E16 mouse SMG, that are highly enriched for Kit and AQP5 positive pro-acinar cells (Nelson et al., 2013). Primary E16 SMG cells are liberated from glands using a combination of enzymatic digestion and manual microdissection. Glands are first manually micro-dissected into large discreet lobes, which are then further micro-dissected into smaller lobule structures with enzymatic digestion. Cell preparations enriched for large epithelial cell clusters or mesenchymal cells are made using limited gravity sedimentation, where the epithelial cell clusters sediment faster than the largely single mesenchymal cells, allowing for removal of the mesenchymal cell-enriched supernatant from the epithelial cell-enriched cell pellet.

Materials

Dissecting microscope with transmitted light base and overhead light

Inverted Tissue Culture Grade Microscope equipped with a digital camera such as a Nikon Eclipse TS100 microscope equipped with a Canon EOS 450D digital camera at 4x (Plan 4x /0.10 NA) or 10x (10x Ph1 ADL/0.25 NA)

Standard tissue culture incubator, humidified at 37°C with 5% CO2

Standard tissue culture benchtop centrifuge

Standard water bath

Standard serological pipets and pipetmen with standard tips

Standard class II biological safety cabinet

Hank’s balanced salt solution (HBSS) (ThermoFisher Scientific)

CD-1 timed pregnant female mice embryonic day 16 (E16) (day 0 is day of plug discovery) (Charles River Laboratories)

Two fine forceps: dumont #5; inox alloy, 0.05mm X 0.02mm (Fine Science Tools)

Collagenase/hyaluronidase (Stem Cell Technologies)

Dispase II (Gibco)

1XPBS (ThermoFisher Scientific)

DMEM:F12, no phenol red (ThermoFisher Scientific) (21041025)

Penicillin 10,000 U/mL / Streptomycin 10,000 μg/ml (ThermoFisher Scientific)

Fetal Bovine Serum (FBS) (ThermoFisher Scientific)

35 mm dishes (Corning)

15 ml conical tubes (Corning)

Collect submandibular glands (SMG) with or without sublingual glands (SLG) from mouse embryos

  1. Following an approved IACUC protocol, remove E16 embryos from 2 timed pregnant females of the desired genotype.

This protocol is designed for cell isolation from 2 litters of CD-1 mice, or about 48 SMG, although the volumes of each reagent can be scaled up or down for limiting or amply gland numbers.

  • 2. Decapitate the embryos heads above the shoulders using sharp scalpels with the embryo laying on its side. Keeping the head on its side, use a sharp scalpel to remove the the lower mandible, slicing between the upper and lower mandible through the back of the neck. Then remove the SMG/SLG with sterile forceps.

Please see (Sequeira, Gervais, Ray, & Larsen, 2013) for complete protocol on harvesting SMG/SLG from smaller E13 embryos, which is similar to harvesting from E16 embryos. As the SMG and SLG are very closely associated, the smaller SLG must be manually separated from the larger SMG and at this step if only SMG cells are desired.

  • 3. Place the SMGs in a 35 mm dish in sterile 1 ml 1XPBS.

  • 4. Optional: Retain a small sample of the gland and chop into small tissue pieces (1 mm diameter) using a sterile scalpel to immunostain in parallel with the organoids to compare marker expression in the native tissue. Fix immediately in 4% PFA in 1XPBS overnight at 4°C. Store in 1 ml 1X PBS at 4°C until ready to stain with cultured organoids.

Enzymatic digestion to liberate epithelial clusters and mesenchymal cells

  • 5. Prepare 1 ml of a 2X collagenase/hyaluronidase solution diluted in 1XPBS.

Make the diluted enzyme solution fresh from a frozen aliquot prior to each experiment.

  • 6. Transfer glands to a 35 mm dish containing 1 ml of 2X collagenase/hyaluronidase solution and place the dish under a dissecting microscope.

  • 7. Use forceps to tease apart glands into lobes; work quickly to tease apart lobes in approximately 15 minutes.

Do not exceed 25 minutes for this step.

  • 8. Add 1 ml dispase (D) stock solution (Cf = 0.8 U/ml) and microdissect lobes to lobules; work quickly to tease apart lobules in approximately 15 minutes.

Note that the addition of dispase causes the lobules to form clumps. Do not exceed 25 minutes for this step.

  • 9. Place the dissected lobules in collagenase/hyaluronidase/dispase enzyme solution in the 35 mm dish with lid to 37°C tissue culture incubator for 30 minutes.

  • 10. Remove dish from the incubator and return to the dissecting microscope.

  • 11. Triturate (10–20x) with P1000 pipette to dissociate tissue fragments into cell clumps.

Under a dissecting microscope, you will see the tissue pieces dissociate into a mixture of cell clusters and single cells, often with the enzyme solution becoming somewhat cloudy from the tissue dissociation. If tissue pieces do not break apart, triturate 10x more. If they still don’t break apart, your enzyme is probably ineffective – repeat steps 7–11 with fresh enzyme.

Separation of epithelial clusters and mesenchymal cells by differential sedimentation

  • 12. Transfer the 2 ml containing the dissociated glands to a 15 ml conical tube. Allow the epithelial-enriched fraction to settle to the bottom of the tube to form a gravity pellet for approximately 5–10 minutes until it appears that the pellet size is no longer increasing and most of the opaque white cloudiness from the cell clumps have settled.

This step is time-sensitive; it is imperative to remove the supernatant after 10 minutes when the epithelial cell clusters have settled and most of the single cells are still in the supernatant. If you pellet too long, there will be more mesenchymal cells in the epithelial-enriched cell fraction.

  • 13. Carefully remove the supernatant with a P1000 pipet, being sure not to disturb the loose epithelial-enriched gravity cell pellet.

  • 14. Place the mesenchyme-enriched gravity supernatant in another 15 ml conical tube and set aside. Add 2 volumes of DMEM/F12 +10% FBS media to the gravity supernatant to stop enzymatic reactions. Keep at room temperature. Mesenchyme cells can be further enriched from this fraction with Support Protocol 2.

  • 15. To the epithelial enriched gravity pellet, add 2 ml of DMEM:F12+10% FBS media to stop enzymatic reactions.

  • 16. Optional: Add 100 μl of DNAse 1 (1 mg/ml) per 1900 μl media (Cf= 0.05 mg/ml) to reduce epithelial cell clumping if needed.

  • 17. Perform two additional gravity sedimentations as in steps 12–13 using 2 ml of DMEM:F12+10% FBS media each time to further enrich the epithelial cell clusters and remove single cells with a P1000 pipet.

  • 18. Pellet the cell suspension by centrifugation for 5 minutes at 450xg; carefully remove the supernatant with a P1000 pipet.

  • 19. Wash cells by resuspending the cell pellet in 2 ml DMEM:F12+10% FBS. Pellet cells for 5 minutes at 450xg and carefully remove supernatant with a P1000 pipet.

  • 20. Resuspend the epithelial-enriched gravity pellet in DMEM:F12+10% FBS.

The resulting epithelial clusters will contain mesenchyme cells. For further enrichment of the epithelial cells refer to Support Protocol 1.

SUPPORT PROTOCOL 1

FURTHER enrichment of epithelial clusters by differential adhesion.

Further enrichment of the epithelial clusters can be achieved by timed differential adhesion followed by differential sedimentation in a centrifuge. In the first step, the single mesenchymal cells that are present in the epithelial-enriched gravity pellet (produced in Basic Protocol 1) are depleted from the epithelial clusters due to their more rapid adhesion to a tissue culture dish relative to the epithelial cell clusters. In the second step, remaining single mesenchymal cells are depleted from the epithelial clusters using 10xg centrifugation for 1 minute. These more purified cell preparations can be used for probing detailed cellular and molecular mechanisms involved in complex organoid development.

Depletion of single mesenchymal cells from the epithelial cluster enriched gravity pellet

  1. Add the resuspended epithelial cell-enriched pellet obtained with Basic Protocol 1 to a 35 mm tissue culture dish.

  2. Incubate at 37°C for 2 hours or until the single cells have adhered to the dish, and the large cell clusters are still rounded and floating, as determined by visual inspection using an inverted tissue-culture microscope.

The timing can be adjusted if needed. If most of the mesenchyme cells are removed, there will be a small number of epithelial clusters attached to the dish. However, if the epithelial cells are allowed to attach for too long, a majority of them will flatten and adhere to the dish.

  • 3. Collect the non-adherent cells in the media using a P1000 pipet and transfer to a 15 ml conical tube.

  • 4. Pellet cells by centrifugation at 450xg for 5 minutes and carefully remove the supernatant.

  • 5. Resuspend the further-enriched epithelial cell clusters in DMEM:F12+10% FBS.

  • 6. Centrifuge the epithelial clusters (ECs) for 1 minute at 10xg.

  • 7. Remove the supernatant and resuspend the cell pellet again in DMEM:F12+10% FBS. The epithelial clusters are largely collected in the pellet, and the single mesenchymal cells are retained in the supernatant.

  • 8. Count the epithelial cell clusters with a hemocytometer, if desired.

As even loading of clusters into a hemocytometer can be difficult, counting cell clusters in a known volume in a dish can be more accurate.

SUPPORT PROTOCOL 2

FURTHER ENRICHMENT OF MESENCHYMAL CELLS

Further processing of the gravity supernatant obtained in Basic Protocol 1 can be performed to remove contaminating epithelial cells to obtain a mesenchyme-enriched cell preparation using sequential gravity sedimentations followed by passage through size exclusion-based cell strainers. The further enriched mesenchyme fraction can be genetically manipulated with lentiviral constructs, as described in Supplementary Protocol 5, and then recombined with epithelium in the presence (Basic Protocol 2) or absence (Alternate Protocol 3) of basement membrane extract to form salivary organoids. The resulting mesenchyme cell population will include multiple mesenchymal cell types: i.e. mesenchymal stem cell-like cells, fibroblasts, endothelial cells, nerve cells, and immune cells. Isolation of mesenchymal cell subsets with with fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS), can be performed on the cells as described elsewhere.(Lee et al., 2017; Moad et al., 2017; Yang, Adam, Ge, Hua, & Fuchs, 2017; Zepp et al., 2017; Zhao et al., 2017)

Additional Materials

70 μm cell strainer (Falcon)

40 μm cell strainer (Fisherbrand)

  1. Perform two additional gravity sedimentations as in steps 12–13 of Basic Protocol 1 to remove residual epithelial cell clusters from the gravity supernatant; retain the supernatant at each step.

  2. Pass the final gravity supernatant sequentially through a 70 μm into a 50 ml conical and then through a 40 μm cell strainer into a 50 ml conical to remove any remaining epithelial cell clusters.

Pass the supernatant through the center of each strainer with a P1000 pipet tip.

  • 3. Pellet cells from the final strained gravity supernatant by centrifugation at 450xg for 5 minutes; carefully remove supernatant and retain the final cell pellet further enriched for mesenchyme cells.

  • 4. Wash the cell pellet with 2 ml 1XPBS; pellet at 450xg for 5 minutes and carefully remove supernatant.

  • 5. Resuspend the mesenchyme-enriched cell preparation in DMEM:F12+10% FBS media.

  • 6. Count cells with a hemocytometer, if desired.

SUPPORT PROTOCOL 3

EMBEDDING EPITHELIAL-ENRICHED CELL PREPARATIONS IN BASEMENT MEMBRANE EXTRACT FOR TIME ZERO CELL CHARACTERIZATION.

Making high quality time zero (tzero) samples is critical for examining the levels and subcellular localization patterns of markers in the original sample and after experimental manipulations. We embed the organoid input cell preparations in Matrigel to create tzero preparations. This makes the sample easy to manipulate and comparable to the format of the organoids for comparison in immunocytochemistry assays (see Support Protocol 5).

Additional Materials

Porous, polycarbonate (Nuclepore) filters (Whatman 110405)

Growth factor reduced Matrigel (Corning) (stored as single-use aliquots at −20C, thawed on ice)

MatTek dishes (MatTek P50G-1.5–14-F)

Fixative Solution (see recipe)

  1. After resuspending the epithelial enriched cell preparation in media for next steps, take one portion of cells (equivalent to the amount of cells used in each sample in Basic Protocol 2) in 5 μl media, mix with 5 μl ice cold Matrigel. Mix quickly by pipetting up and down and immediately plate onto the center of the Nuclepore filter.

Matrigel mixtures should be kept on ice. Resuspend the Matrigel-cell mixture between platings. The use of pre-chilled tips that are also kept on ice is recommended.

  • 2. Allow Matrigel to solidify for 20 minutes at 37°C. If properly stored as single-use aliquots, Matrigel should solidify within this time-frame.

  • 3. Fix samples for 20 minutes with 4% paraformaldehyde (PFA) in 1XPBS at room temperature.

The samples should be fixed as soon the Matrigel has formed a gel to minimize Matrigel-induced outside-in signaling. Other inert gels can be used to embed the cells or the cells may be cultured on coverslips but note that their response to immunostaining may differ from the organoids embedded in Matrigel and culture may change the cells’ properties.

  • 4. Store the samples at 4°C in 1XPBS until the organoids are ready to be analyzed.

Later, perform ICC on tzero cells at the same time as the organoids (See Support Protocol 4).

BASIC PROTOCOL 2

3D CULTURE OF COMPLEX SALIVARY ORGANOIDS IN BASEMENT MEMBRANE EXTRACT.

Epithelial cell clusters can be grown in 3D matrices that enable cell self-organization to form organoids. To make 3D organoids from epithelial cell clusters (produced from Basic Protocol 1), cells in media are mixed with basement membrane extracts (Matrigel or 3D laminin-111), and cultured on top of porous polycarbonate Nuclepore filters floating on media in a glass-bottomed microwell dish (MatTek). Importantly, we determined that mesenchyme cells are critical for organoid formation (Hosseini et al., 2018). The ECs isolated in Basic Protocol 1 contain a sufficient number of mesenchymal cells to support formation of complex salivary organoids with robust AQP5 expressing pro-acinar cell differentiation. If the ECs are further enriched, as described in Support Protocol 1, then mesenchyme cells must be added to the organoid assay. Growth factors and pharmacologicals can be added to the culture set up to test their effects. We determined that inclusion of FGF2 is essential for elaboration of complex salivary organoids containing robust AQP5 positive secretory pro-acinar cell differentiation (See Fig. 2C) (Hosseini et al., 2018). We routinely use simple media containing 100 ng/ml of FGF2 to produce complex salivary organoids that express high levels of membrane-localized AQP5 or simple media containing 100 ng/ml EGF to produce spheroids that do not branch or express AQP5. (see Fig. 2C) (Hosseini et al., 2018).

Figure 2: Formation of Proacinar Organoids in Matrigel Requires FGF2 and Primary Mesenchyme.

Figure 2:

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(A) Schematic of organoid culture, as described in Basic Protocol 2. In Alternate Protocol 1, a mesenchyme cell preparation is mixed with the ECs in basement membrane extract at the time of culture set up. (B) Brightfield images of enriched EC (prepared as in Basic Protocol 1) and grown in Matrigel in media containing FGF2 (as described in Basic Protocol 2). Branched organoids formed within 7 days. (C) ICC (preformed as described in Support Protocol 5) and confocal imaging revealed AQP5+ buds formed in ECs grown in the presence of FGF2 but not EGF. Markers: EpCAM (epithelial, green), AQP5 (proacinar/acinar, red), and vimentin (mesenchyme, cyan) with DAPI staining (nuclei, blue). (D) Schematic of co-culture, as described in Alternate Protocol 2, as reported in Hosseini et al., 2018. (E) Enriched ECs prepared as described in Support Protocol 1. (F) Further enriched ECs generated smooth spheroids lacking end buds. Scale bar = 50 μm. N = 3 experiments. EC = epithelial cluster, Mes = Mesenchyme

Materials

Standard tissue culture incubator, humidified at 37°C with 5% CO2

Standard 37°C water bath

Standard serological pipetmen with standard tips

Standard class II biological safety cabinet

Ice

Tissue Culture Grade Microscope equipped with a digital camera such as a Nikon Eclipse TS100 microscope equipped with a Canon EOS 450D digital camera at 4x (Plan 4x /0.10 NA) or 10x (10x Ph1 ADL/0.25 NA)

1.7 ml microfuge tubes (Axygen)

Polycarbonate (Nuclepore) filters (Whatman 110405)

Growth factor reduced Matrigel (Corning)

Cultrex 3D culture matrix laminin-111 (Trevigen)

MatTek dishes (MatTek P50G-1.5–14-F)

Simple media (see recipe)

Recombinant murine Fibroblast Growth Factor (FGF2) (Peprotech)

Recombinant human Epidermal Growth Factor (EGF) (Peprotech)

Fixative Solution (see recipe)

Sterile fine-tipped scissors

Embed enriched epithelial cell clusters in basement membrane preparation

  1. Place a Nuclepore filter (shiny side up) on top of a 200 μl drop of media in the center well of a MatTek dish for each condition in the experiment. Cut a small notch (2–3 mm deep) in the edge of the filter for reproducible orientation of the filter during bright field imaging, if desired.

  2. Resuspend the enriched SMG ECs in 5 μl of simple media containing 100 ng/ml FGF2 per sample.

We have used 350–1000 epithelial cell clusters (from 1.25–2.5 SMG) per filter for generation of complex salivary organoids with robust AQP5 expression, with three replicates per condition. Media can contain other growth factors or bioactive compounds as needed to investigate effects on organoids, see Hosseini-18 for examples.

  • 3. For each filter, mix enriched ECs in 5 μl media with 5 μl of the ice-cold growth factor reduced Matrigel (or 3D laminin-111) and pipet on top of the Nuclepore filter with gentle spreading of the drop starting from the center of the filter to cover 20–60% of the filter area.

Basement membrane preparations should be kept on ice at all times to prevent polymerization before culture, and cell-Matrigel suspensions should also be kept on ice before plating. Be sure that the Matrigel does not reach the edge of the filter or the cut notched area.

  • 4. To be able to compare the properties of the organoids to the starting cell populations, set up the desired number of replicates of tzero samples (see Support Protocol 3).

Form organoids

  • 5. Culture the cell preparations in a tissue culture incubator for 7 days to form proacinar organoids.

We routinely culture cells for 7 days to obtain complex salivary organoids that express robust, membrane-localized, AQP5.

  • 6. Change the media during the culture period using a P200 pipet tip to remove the media from under the filter. Carefully add new media containing the appropriate additives under the filter using a P200 pipet tip.

We routinely change the media at day 4. It is helpful to use sterile forceps to gently lift the filter up with one hand and apply new media underneath it with the other hand.

  • 7. Optional---capture brightfield images daily using a standard phase contrast tissue culture microscope with a digital camera attached. Use the notch in the filter to reproducibly position the filter in the same orientation.

  • 8. At the end of the culture period, prepare the samples for ICC (Support Protocol 5) or other end point assay.

ALTERNATE PROTOCOL 1

3D culture of complex salivary organoids using further enriched SMG epithelial cell clusters with mesenchyme cell ADD-BACK.

Culture of the more purified epithelial clusters having very few associated mesenchyme cells (from Support Protocol 1) did not form complex salivary organoids expressing high levels of membrane-localized AQP5 (Fig. 3A,C) (Hosseini et al., 2018). Here we report, a method for add-back of enriched preparations of salivary mesenchyme (from Support Protocol 2) to the 3D cultures which rescues complex salivary organoid formation and membrane-localized AQP5 expression (Fig. 3C).

Figure 3: Complex FGF2-Dependent Salivary Proacinar Organoids Require Mesenchymal Cells.

Figure 3:

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(A) Epithelial clusters that include mesenchyme cells (produced by Basic Protocol 1) when embedded in Matrigel and grown in media containing FGF2 (as described in Basic Protocol 2) form branched proacinar organoids expressing AQP5 while further enriched ECs produced with Support Protocol 1 and grown under the same conditions do not. Images are widefiled subjected with ICC to detect: AQP5 (proacinar epithelium, red), vimentin (mesenchyme, cyan) with DAPI (nuclei, blue). (B) Quantitative analysis demonstrates AQP5 is retained with FGF2 and basement membrane only when mesenchyme is also present. (C,D) Further enriched ECs (produced using Support Protocol 1) that do not form proacinar organoids when cultured in Matrigel in media containing FGF2 (as in Basic Protocol 2) form branched proacinar organoids expressing AQP5 when enriched mesenchyme (prepared as in Support Protocol 2) is added back. N=3 experiments for each condition. Scale bars: 50 μm.

Mix ECs with mesenchyme cell preparation.

  1. Count epithelial clusters from the more highly enriched epithelial progenitor preparation (Support Protocol 1) using a hemocytometer or perform cell counts from cells in a dish that have been allowed to settle. The amount of time for cell counting should be minimized to prevent ECs from sticking to each other or to the plate.

  2. Mix EC with 4.0 X 104 to 2.0 X 105 mesenchyme cells (from 0.25 to 1.25 SMG) to achieve a 5 μl cell volume per sample.

  3. Set up 3D organoid cultures, as in Basic Protocol 2, mixing mesenchyme cells with the ECs in step 2.

Use only enriched ECs from Support Protocol 1to avoid contaminating mesenchyme cells provided by the ECs.

ALTERNATE PROTOCOL 2

CO-CULTURE OF SMG EPITHELIAL CELL CLUSTERS WITH A MESENCHYME CELL FEEDER LAYER

To produce complex salivary organoids with robust AQP5 expression in the absence of an exogenous basement membrane preparation, enriched mesenchyme cells (Support protocol 2) can be seeded onto a floating polycarbonate Nuclepore filter to create a feeder layer to support the epithelium in a co-culture assay. ECs from Basic Protocol 1 or further enriched ECs from Support Protocol 1 can be used in the co-culture assay. However, it is recommended that the further enriched ECs from Support Protocol 1 be used in the co-culture assay if genetically manipulated mesenchyme cells are used to attribute specific contributions to specific cell populations. Co-culture of mesenchyme cells transduced with an FGF2 shRNA allowed us to demonstrate that FGF2-expressing mesenchyme cells are required to sustain AQP5 expression in the organoids (see Fig. 4C,E ) (Hosseini et al., 2018).

Figure 4: Lentiviral delivery of FGF2 shRNA to mesenchyme demonstrates requirement for FGF2 expression in mesenchyme in salivary organoids formed by co-culture with mesenchyme feeder layer.

Figure 4:

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(A) Schematic of an epithelial and mesenchyme co-culture for 7 days, as described in Alternate Protocol 2. (B) Mesenchyme cells prepared as described in Support Protocol 2 were transduced with a lentivirus expressing RFP and imaged live by widefield and fluorescent imaging after 72 hours. (C) Mesenchyme prepared as described in Support Protocol 2 and transduced with lentivirus expressing an FGF2 shRNA or control shRNA (as described in support protocol 4) was combined with epithelium, as described in Support Protocol 2, grown for 7 days, and subjected to ICC, as described in Support Protocol 5. ICC revealed association of vimentin-positive mesenchyme with the epithelial cells within the organoid. Markers detected: EpCAM (epithelial cells, green), AQP5 (proacinar cells, red), and vimentin (mesenchyme cells, cyan) with DAPI (nuclei, blue). Quantification of confocal imaging revealed: (D) a slight decline in the epithelial population and (E) significant loss of proacinar differentiation. (F) Knockdown was quantified by ELISA assay to detect FGF2 from cell extracts, as reported in Hosseini, et al., 2018. *p < 0.05 and **p < 0.01. (Student’s t-test for for E and D and one-way ANOVA with Tukey post-hoc test for F). Scale bar = 50 μm. N = 3 experiments

Form the mesenchymal cell feeder layer

  1. Resuspend the non-manipulated enriched mesenchyme cells (Support Protocol 2) or lentiviral construct transduced cells (Support Protocol 4) in 10 μl of DMEM:F12+10% FBS media per sample.

We routinely use 1.1 X 106 mesenchyme cells (equivalent of 5 SMGs) to make the feeder layer. Plate the mesenchyme onto the filter and culture in the 37°C tissue culture incubator for 4 hours to allow the mesenchyme to adhere, flatten, and form a monolayer.

Form epithelial-mesenchymal co-culture

  1. Add the epithelial cell preparation in 5 μl of DMEM:F12+10% FBS media directly onto the mesenchyme cell feeder layer and return to the 37°C incubator. We use 350–1000 epithelial cell clusters (from 1.25 to 2.5 E16 SMG) per filter.

  2. Grow in tissue culture incubator for 7 days to obtain complex salivary organoids, changing the media at day 4 by carefully pipetting the media from under the Nuclepore filter and replacing it with fresh media.

  3. At the end of the culture period, remove the media from under the filters, and fix the organoid co-cultures by addition of 200 ul of 4% paraformaldehyde in PBS at 4°C overnight.

  4. Optional: image filters daily using a bright field microscope with an integrated digital camera to monitor culture growth.

The co-cultures of epithelium and mesenchyme are not as three dimensional as the cultures grown in basement membrane extract.

SUPPORT PROTOCOL 4

LENTIVIRAL INFECTION OF SALIVARY MESENCHYME FOR INCORPORATION IN ORGANOIDS

We have used lentivirus infection of the salivary mesenchyme to characterize molecular mechanisms governing organoid formation and function in the Matrigel organoid assays (Basic Protocol 2) and co-culture assay (Alternate Protocol 2). Mesenchyme cells transduced with lentivirus particles can be characterized for exogenous protein expression or efficacy of shRNA-mediated knockdown. Lentiviral transduced cells can be used together with epithelial clusters in the organoid assay (Basic Procotol 2) or Alternate Protocol 1 or 2. Using this technique, we knocked-down FGF2 in the mesenchyme to demonstrate that expression of FGF2 by the mesenchyme is required to support proacinar differentiation of salivary organoids (see Fig. 4C) (Hosseini et al., 2018).

CAUTION: NIH guidelines suggest that replication-incompetent lentiviral particles be handled as Risk Group-Level 2 (RGL2) and handled in a BSL2 facility. Additional precautions may be required based upon local, state, or country regulations. When working with lentiviral particles obtain Institutional Biosafety Committee approval at your institution. In general, use appropriate personal protective equipment, clean all contaminated surfaces by exposure to 10% bleach for 20 minutes and dispose of all solutions containing lentivirus as biohazard waste.

Additional Materials

Standard class II biological safety cabinet in a BSL2 facility

MISSION pLKO.1-puro Non-Mammalian shRNA Control Transduction Particles (Sigma SHC-002V)

MISSION pLKO.1-puro shRNA Transduction Particles (for FGF2, FGF2 shRNA lentivirus particles #SHCLNV-NM-008006; TRCN00000–67283)

Gelatin (Fisher Scientific)

Polybrene (Millipore Sigma)

Lentiviral infection

  1. Plate mesenchyme-enriched cell preparation cells (see Support Protocol 2) in simple media onto a gelatin-coated 10 cm dish.

  2. Culture for 1–3 days in tissue culture incubator, changing the media on day 1.

  3. Trypsinize the cells from the tissue culture plate using standard methods.

  4. Per sample, suspend 3 X 105 mesenchyme cells in simple media.

  5. Dilute a 106 TU/ml lentivirus preparation into the cell suspension.

We have found a 1:5 to 1:10 dilution of the lentivirus preparation gives at least 80 percent infection of the primary mesenchyme when infected with RFP expressing virus and robust knockdown of FGF2 by ELISA when infected with the MISSION FGF2 shRNA lentivirus.

  • 6. Add polybrene so final concentration is 8 μg/ml polybrene in 1.5 ml solution.

  • 7. Transfer cells containing viral particles to a single well of a 6-well tissue culture plastic plate covered in gelatin.

  • 8. Remove media containing lentiviral particles after 24–72 hrs and replace with simple media.

  • 9. Culture for 3–14 days in tissue culture incubator, changing the media at least every three days.

Maximum expression should be achieved by 72 hrs. Antibiotic selection can be used to enrich for lentiviral transduced cells.

  • 10. Trypsinize the cells from the tissue culture plate using standard methods.

  • 11. Resuspend in desired volume of media and count cells using a hemocytometer.

  • 12. Use cells in a co-culture assay with basement membrane extract (Alternate Protocol 1) or in the absence of basement membrane extract (Alternate Protocol 2).

SUPPORT PROTOCOL 5

QUANTITATIVE IMMUNOCYTOCHEMISTRY (ICC) ANALYSIS OF SALIVARY ORGANOIDS.

Examination of expression levels and spatial distribution of cell type-specific markers and other molecular markers of interest can be obtained with whole mount ICC of organoids. Although widefield fluorescent imaging can be used to image the 3D cell preparations and organoids, the clearest images are obtained using laser scanning confocal microscopy. Image processing then involves either counting of marker positive cell clusters or organoids, or quantification of marker-positive areas using thresholded and calibrated images. Image processing is followed by graphing and statistical analysis. Please see other references for detailed discussion of image acquisition and analysis (Oberholzer, Östreicher, Christen, & Brühlmann, 1996). Importantly, quantification allowed us to demonstrate that inclusion of FGF2 promoted expansion of residual vimentin-positive mesenchyme in the epithelial-enriched gravity pellet concomitant with emergence of epithelial buds with robust, membrane-localized AQP5 expression indicative of secretory pro-acinar cell differentiation in the complex salivary organoids (Hosseini et al., 2018). Importantly, comparison of the cultured organoids relative to the tzero cell preparations allows for quantitation of marker levels relative to the starting input cell preparations, and assessment of native subcellular distribution of proteins.

Additional Materials

Washing Solution (see recipe)

Permeabilization Solution (see recipe)

Bovine Serum Albumin (BSA) (Sigma)

Donkey serum (Jackson ImmunoResearch) - Store aliquots at −20°C

Anti-Aquaporin 5 (Alomone)

Anti-Vimentin (Sigma)

EpCAM-FITC (eBiosciences)

Fluorophore conjugated secondary antibodies including Cyanine- or Alexa dye-conjugated AffiniPure F(ab′)2 fragments (donkey anti-mouse IgM or anti-rabbit IgG), as required to detect primary antibodies (Jackson Immunoresearch)

DAPI (Sigma)

Fluorogel with Tris buffer mounting media (Electron Microscopy Sciences)

SecureSeal imaging spacers (Grace Biolabs))

Microscopic glass slides (Fisher Scientific)

Coverslips (Thermo Scientific)

Nail polish

Slide books for slide storage

Standard multichannel widefield and/or confocal microscope.

Fix and permeabilize cells

  1. Fix cell or organoid preparations on Nuclepore filters in MatTek dishes with 200 μl of fixative solution for 20 minutes at room temperature.

If the fixative is added underneath the filter, the cells/organoids will typically stay attached to the filter for ease of manipulation in subsequent steps.

  • 2. Wash 3X with 200 μl 1XPBS-T for 10 minutes.

  • 3. Permeabilize with 0.4% Triton-X-100 in 1XPBS for 20 minutes.

Nuclear antigens may require permeabilization for up to twice as long.

  • 4. Wash with 200 μl 1XPBS-T for 10 minutes.

Block non-specific binding

  • 5. Block with 200 μl 1XPBS-T+3%BSA+10% donkey serum at room temperature for 1 hour or overnight at 4°C.

Antibody incubations and washes

  • 6. Incubate with primary antibodies diluted in 1XPBS-T+3%BSA overnight at 4°C.

Overnight incubation is important for primary antibodies in 3D cultures.

  • 7. Wash 4X with 200 μl 1XPBS-T for 10 minutes.

  • 8. Incubate with fluorophore-conjugated secondary antibodies diluted in 1XPBS-T+3%BSA-5% donkey serum, overnight at 4°C. Include 10 μg/ml DAPI to stain nuclei.

  • 9. Wash 3X with 200 μl 1XPBS-T for 10 minutes.

Mounting coverslips for imaging

  • 10. Prepare slides with imaging spacers by exposing sticky side and carefully sticking onto the slide and then removing the cover to expose the other sticky side of the spacer.

  • 11. Carefully place the filter into the hole created by the imaging spacer.

  • 12. Pipet 40 μl of mounting media onto the filter in the hole.

  • 13. Cover the filter with a glass coverslips.

  • 14. Allow mounting media to harden and dry for 1hr.

  • 15. Optional: Seal coverslips with nail polish to prevent evaporation.

  • 16. Store slides at −20°C until use and after imaging.

Imaging and quantification

  • 17. Perform multichannel fluorescence imaging using the same laser configuration settings in each channel for all samples to allow quantitative comparisons of the images.

  • 18. To quantify marker positive cell clusters or organoids, manually count the number of clusters or organoids using the DAPI channel and then manually count the number of clusters or organoids that are positive for the marker of interest. We previously required the organoids to have at least 30% of the area expressing AQP5 to be defined as being AQP5+.

  • 19. To quantify marker expression, use Image J to calculate marker positive areas of color thresholded images relative to DAPI-stained nuclei.

  • 20. For quantitative comparisons, capture at least 5 images of each sample and perform at least 3 independent experiments.

REAGENTS AND SOLUTIONS

Collagenase/hyaluronidase (C/H)

10x stock solution (Stem Cell Technologies, #07912). Make single use aliquots and store at −20°C. For SMG dissociation, make 2X solution in 1XPBS.

Dispase I Stock Solution (1.6 U/ml)

Add 0.5g of dispase I to 50 ml milliQ water. Make 1 ml single aliquots and store at −20°C.

Fixative Solution

To make a 4% paraformaldehyde (PFA) solution with 5% sucrose, add 10 ml of 16% PFA (Electron Microscopy Sciences) to 4 ml of 10XPBS with 2 g sucrose in a 50 ml conical tube with milliQ water to make a 40 ml total volume. Cover the tube with aluminum foil to protect from light. Store at 4°C and use for no longer than 2 weeks. Fix for 20 minutes at room temperature or overnight at 4°C depending upon the sample.

Other fixation methods can be used, and we routinely use ice-cold methanol fixation for 18 minutes without subsequent detergent permeabilization to detect ductal cytokeratins that require this fixation method. All fixations using methanol should be done at −20°C.

Washing Buffer (1xPBS-T)

0.5% Tween-20 (Sigma) in 1XPBS

Permeabilization Solution

0.4% Triton X-100 (Sigma) in 1XPBS

Simple Media

50 ml fetal bovine serum (FBS)

450 ml DMEM:F12

5 ml penicillin-streptomycin

Store at 4°C

Recombinant bFGF Stock Solution, 100 μg/ml

Resuspend lyophilized recombinant basic fibroblast growth factor (bFGF; Peprotech) in sterile 0.2% BSA solution to a final concentration of 100 μg/ml. Make aliquots and store at −20°C. Avoid repeated freezing and thawing.

Recombinant EGF Stock Solution, 100 μg/ml

Resuspend lyophilized recombinant epidermal growth factor (EGF; Peprotech) in sterile 0.2% BSA solution to a final concentration of 100 μg/ml. Make aliquots and store at −20°C. Avoid repeated freezing and thawing.

COMMENTARY

Background Information

Reconstructing the third dimension in cultured cells to recapitulate cell-cell signaling that occurs in organs in vivo is of increasing interest in recent years. The process of generating 3D assemblies of cells that self-organize to generate structures resembling mini organs is referred to as organoid technology (Bissell, 2017; Clevers, 2016; M. Simian et al., 2001; Marina Simian & Bissell, 2017). The term “organoid” was first used to describe branching 3D structures generated from clusters of mammary gland cells (M. Simian et al., 2001), but the term has now been expanded to encompass multiple techniques for forming 3D organ-like structures, ranging from primary explants of tissue fragments to epithelial-mesenchymal co-cultures to clonal derivatives of primary epithelial stem cells (Shamir and Ewald, 2014; Kretzschmar and Clevers, 2016; Simian and Bissell, 2017). Organoids can be used to model normal developmental processes (Clevers, 2016) and can be manipulated to examine the contribution of specific signaling pathways without the need to generate transgenic animals. Organoids derived from stem cells can be induced to differentiate down specific lineages by addition and/or withdrawal of specific factors often in the presence of ECM extracts (reviewed in (Akkerman & Defize, 2017; Fatehullah, Tan, & Barker, 2016; Huch & Koo, 2015; Kretzschmar & Clevers, 2016)).

Studying cell-cell interactions in organoid development has become a useful tool for understanding mechanisms leading to tissue regeneration (Shamir & Ewald, 2014). Mixed cell populations from multiple tissue sources formed organoids capable of self-organizing and differentiating in culture and during in vivo implantation, as has been shown that many organoids contain mesenchyme, particularly embryonic and induced pluripotent stem cell derived-organoids (Dye et al., 2015; Hegab et al., 2015; McQualter, Yuen, Williams, & Bertoncello, 2010; Shamir & Ewald, 2014; Spence et al., 2011; Teisanu et al., 2011; Asai et al., 2017; Mondrinos, Koutzaki, Lelkes, & Finck, 2007; Nikolić & Rawlins, 2017; Takebe et al., 2013, 2017; Tan, Choi, Sicard, & Tschumperlin, 2017). Fibroblasts have been shown to be critical in providing niche factors for stem cells and organoid survival in the intestine (Pastuła et al., 2016). Recent studies in the salivary gland have indicated that stem cells isolated from adult organs can be expanded and differentiated to generate organoids capable of restoring gland function in vivo (Maimets et al., 2016; Nanduri et al., 2014). However, the role of epithelial-mesenchymal interactions in promoting organoid formation and maturation has not been investigated in the salivary gland.

An important limitation of many organoids is that the differentiated cell distributions may not include all of the important cell types native to the tissue or organ, and the differentiated cells often do not differentiate to full maturity. For salivary gland epithelial explant cultures and organoids, ductal cell expansion and differentiation are commonly observed, however robust secretory cell differentiation has been difficult to achieve. We here describe methods for creating complex salivary organoids that mimic native tissue architecture containing salivary mesenchyme cells that support robust secretory pro-acinar cell differentiation from salivary epithelial progenitor cells. Use of these complex salivary organoids will facilitate elucidation of cellular and molecular mechanisms by which the heterotypic cell populations of the salivary gland interact to create and sustain organ function, which can then be exploited for improved therapeutic strategies to restore salivary function in patients suffering from salivary gland deficiencies.

Critical Parameters and Troubleshooting

In Basic Protocol 1, the enzyme concentration and incubation time are critical parameters for SMG dissociation. High enzyme concentrations or prolonged incubation in enzymatic solution results in smaller epithelial clusters which are harder to separate from mesenchyme cells as well as more single cells that do not incorporate into organoids. Epithelial clusters that have at least 10–20 cells in them can be separated from the gravity supernatant as a gravity pellet and will form organoids. Timing of the gravity sedimentation is also essential, as eventually all cells will pellet. Both the epithelial clusters and mesenchyme cell preparations obtained from Basic Protocol 1 are complex cell populations containing some cells from the other cellular compartment.

It is important to note that the centrifugation speed is critical to obtain an enriched epithelial cell preparation containing mesenchyme cells from Basic Protocol 1. Pelleting the epithelial preparation at a speed lower than 450xg leads to reduced accumulation of mesenchyme cells in the epithelial cell cluster preparation, reducing organoid formation in Basic Protocol 2 with reduced branching and expression of AQP5. This property, however, can be exploited for further enrichment of the epithelial clusters, and in our experience, differential adhesion of epithelial clusters followed by differential centrifugation in Support Protocol 3 gives more consistent mesenchyme depletion than either method alone.

Cell density of both the epithelium and mesenchyme cells are essential factors for promoting salivary gland organoid formation. The number of epithelial clusters seeded initially in the basement membrane preparation determines whether organoids with AQP5+ budded structures will be generated (Basic Protocol 2). In our experiments performed using Basic Protocol 1, optimal organoid formation occurs with 350–1000 epithelial clusters. With lower numbers of epithelial clusters, non-branched epithelial clusters will remain, although supplementation with extra mesenchyme cells can partially rescue complex organoid formation with lower densities of epithelium. With higher numbers of epithelial clusters, the clusters will tend to aggregate and all of the AQP5+ branches protrude only from the outer edgesof the large aggregates. In the 3D organoids, mesenchyme is also essential for organoid formation. With the further enriched epithelial cluster preparation in Support Protocol 1, it is imperative to add back at least 4.0 X 104 mesenchyme cells to achieve branched AQP5+ organoids. In the co-culture assay (Alternate Protocol 2), the high number of mesenchyme cells seeded as a feeder layer contributes to effective formation AQP5+ organoids, although the epithelial survival in the co-culture assay (Alternate Protocol 2) is not as robust as in the 3D assay with basement membrane preparations (Basic Protocol 2). SeeHosseini, et al., 2018 for additional details.

The organoids can be analyzed with many methods other than the ICC protocol we describe here. In this protocol, the durations of fixation and antibody incubation are critical factors for obtaining good ICC in Support Protocol 4, and these steps may need to be optimized for antigens other than those described here. To assess organoid formation, we find widefield live brightfield images to be helpful. Differentiation can be assessed at a general level with a 5X or 10X with confocal imaging being necessary for high magnification imaging from 20X to 60X.

Statistical Analyses

For all experiments, we use triplicate conditions with a minimum of three independent experiments, and two-way ANOVA with post-tests for multiple comparisons to determine if a given sample is significantly different from any of the other samples. For statistical analysis of image data acquired following ICC in Support Protocol 5, Student’s two-tailed t-tests should be used for comparisons of two conditions to test if one condition is significantly different from the other. For a more in-depth discussion of appropriate statistical analysis, please see additional references (Abràmofff, Magalhães, & Ram, 2005; Hosseini et al., 2018).

Anticipated Results

In Basic Protocol 1, enrichment of epithelial cells in the gravity pellet and mesenchyme cells in the gravity supernatant is assessed by ICC, as shown in Fig. 1 and detailed in Hosseini et al., 2018. The Support Protocols 1 and 2 provide methods for further enrichment of the epithelium and mesenchyme to allow characterizations of molecular and cellular mechanisms by which the heterotypic cell interactions promote organoid formation.

Figure 1: Dissociation and Enrichment of Embryonic Day 16 (E16) SMG Mesenchyme and Epithelium.

Figure 1:

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(A) E16 SMG were subjected to enzymatic treatment using collagenase/hyaluronidase/dispase (C/H/D) to generate lobules followed by incubation and trituration to separate the epithelial clusters (ECs) and mesenchyme cells through three sequential gravity sedimentation steps (Basic Protocol 1). (B) Brightfield images showed the morphology of the gland before enzymatic treatment, and after enzyme treatment to isolate lobules. After processing and gravity sedimentation, the ECs are enriched in the gravity pellet, and the mesenchyme cells are enriched in the gravity supernatant. (C) ICC (as described in Support Protocol 5) and confocal imaging revealed that AQP5 is apically localized in the acini in pieces of the whole gland before enzymatic digestion, in lobules, and in epithelial clusters from the gravity pellet prepared as time zero samples (as described in Support Protocol 3). Gravity supernatant enriched for mesenchymal cells includes cells that express vimentin. Markers: EpCAM (epithelial, green), AQP5 (proacinar/acinar, red), and vimentin (mesenchyme, cyan) with DAPI staining (nuclei, blue). Individual channels are shown at right from boxed areas, as reported in Hosseini et al., 2018. Scale bar = 200 μm for (B) and 50 μm for (C). N = 3 experiments. Mes = mesenchymal cells, EC = epithelial clusters, Supe = supernatant.

Basic Protocol 2 describes culture conditions for complex salivary organoid formation with robust, membrane organized AQP5, as shown in Fig. 2. The complex 3D salivary organoids generated after 7 days have an average diameter of 220–250 μm in the presence of basement membrane extracts, mesenchyme and FGF2. These organoids form complex budded structures expressing membrane-localized AQP5, resembling the in vivo structure of the gland. Epithelial clusters cultured with EGF or no mesenchyme in the presence of basement membrane extracts form spheroids lacking AQP5 expression. The alternate and support protocols provide methods to facilitate independent manipulation and genetic modification of the heterotypic cell populations that synergize for complex salivary organoid formation to enable characterization of molecular and cellular mechanisms involved in organoid formation, which may be exploited for future therapeutic interventions for restoration of salivary gland function. Support Protocol 5 details the methods for quantitative ICC that are important for determining the degree to which the resulting organoid populations resemble the intact salivary gland.

Time Considerations

The entire Basic Protocol 1 and takes 2–3 hours, and Basic Protocol 2 takes about 30 minutes. Co-culture with mesenchyme as a feeder layer requires an additional 4 hours to create a feeder layer.

Lentiviral transduction of mesenchyme takes approximately 3 days. Co-culture of lentiviral-transfected mesenchyme with epithelial clusters takes from 10–14 days. The organoids will form over a period of days with robust expression of AQP5 detectable within 4–5 days and is more widespread and organized by day 7.

ICC of samples takes about 2 days and imaging plus analysis of data may take another 2–5 days depending on the number of samples.

Significance Statement.

While organoid technologies are enabling detailed characterization of mechanisms and heterotypic cell-cell interactions promoting morphogenesis, differentiation and retention of diverse functional cell types in vitro, robust differentiation of salivary secretory acinar cells from epithelial progenitor cells in vitro has not yet been achieved. We here describe methods for creating complex salivary organoids that mimic native tissue architecture containing embryonic salivary mesenchyme cells that support robust secretory pro-acinar secretory cell differentiation from embryonic salivary epithelial progenitor cells. These salivary organoids provide a system to explore requirements for signals provided by salivary mesenchyme in promoting morphogenesis and differentiation of secretory salivary epithelium from embryonic progenitor cells. Future use of organoids will facilitate improved regenerative medicine approaches to restore salivary gland secretory function.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health (R01 DE022467, R56 DE2246706, R21 DE027571and, C06 RR015464, and R01 GM5154017) and the University at Albany, SUNY. The authors thank Drs. James Castracane and Nathaniel C. Cady for use of the Leica SP5 LSCM which was obtained through a grant from the National Science Foundation [DV10922830].

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