TITLE: Establishment of craniofacial exocrine gland organoid magnetic bioassembly platforms as aging multi-omic signatures
AUTHORS: Teerapat Rodboon, Glauco  Souza, Apiwat   Mutirangura, Joao   N. Ferreira

[DESCRIPTION]
In the last decade, relevant biotechnology advances took place in the biofabrication of craniofacial exocrine gland organoids mimicking lacrimal and salivary glands. Though certain challenges still remain not only due to the lack of protocols for organoid reproducibility but also towards the scarcity of methodologies for creating preclinical disease models with aging multi-omic signatures. Previously, our research group successful developed three-dimensional (3D) bioassembly technologies towards the generation of functional epithelial gland-like organoids via magnetic 3D bioprinting platforms (M3DB). To meet the needs of our aging Asian societies, a next step was taken to design consistent M3DB protocols for bioengineering organoid models with aging molecular and pathological features for lacrimal glands (LG) and salivary glands (SG). Herein, we established a feasible step-by-step protocol for producing both LG and SG organoids using M3DB platforms. Such a protocol provided reproducible preliminary outcomes resembling LG/SG organoids. Both acinar and ductal epithelial compartments were prominent (21 4.32% versus 42 6.72% of total cells, respectively), and could be clearly identified in these organoids. Meanwhile, these can be further developed into aging signature models by inducing cellular senescence via chemical mutagenesis. The generation of senescence-like organoids is our ultimate milestone aiming towards high throughput applications for drug screening and discovery and gene therapy investigations to reverse aging.

[STEPS]
SECTION: Mechanical and enzymatic primary cell dissociation from craniofacial exocrine glands
117. Gland dissection and mechanical tissue dissociation 
1.2. Clean the porcine head with sterilized water to remove any debris from the skin before disinfecting it with 0.5 % (v/v) peracetic acid solution for 15 min.

1.3. Gently wash with 1 L of sterilized reverse osmosis (RO) water for 5 times and dry the skin by applying tissue paper.
1.4. Disinfect the skin around the eyes (for the LG) and at the angle of the mandible (for the SG) with 70 % (v/v)  ethanol followed by 1 % (w/v)    and dissect the glands (Figure 1A).

1.5. Collect the glands in a  50 cm Petri dish (Figure 1B) and disinfect with 70 % (v/v)  ethanol. To protect the dry out of tissue, covering the gland with 2 mL of collection media.


1.6. Remove peripheral fat and connective tissue by using precision forceps and scissors (Figure 1C) before sectioning the tissue with a scalpel into   to    pieces (Figure 1D).

1.7. Transfer tissue into a 50 mL collection tube and wash with 20 mL collection media for 3-5 times or until the solution is clear.
1.8. Keep the tissue in 20 mL collection media at 4 °C to7 °C for 360 min to480 min.
 

1. Mechanical and enzymatic primary cell dissociations from craniofacial exocrine glands
2. Aging Organoid Establishment with Magnetic Bioassembly
SECTION: Gland dissection and mechanical tissue dissociation
1.1. Lacrimal gland (LG) and salivary submandibular gland (SG) are dissociated from a 3 to 5 month-old porcine head. The head is carried inside an ice-containing box and delivered to the laboratory within 480 min  after animal sacrifice.

1.10. Mince the tissue using a surgical curved scissor into an apple sauce-like slurry paste (Figure 1D) and transfer the slurry into a 15 mL conical tube with a pre-wet 2.5 mL disposable Pasteur pipette.
1.11. Add 10 mL of washing buffer and pipette up and down by using a pre-wet 10 mL serological pipette.

 

1.12. Let the tissue settle at the bottom of the tube by gravity for 3 min and remove the supernatant using a pre-wet 10 mL serological pipette.
 


1.15. Add 2 mL (for 200 mg of tissue) of digestion buffer into a tissue fragment and gently mix well by vortexing. Make sure that no tissue fragment sticks on the lateral walls of the tube after vortexing.

1.14. Centrifuge the tissue slurry at 1000 x g, 5 min and carefully discard the supernatant.

1.13. Repeat steps    to   for 3-5 times until the supernatant is clear.

1.16. Wrap the cap and neck of the tube with parafilm to minimize contamination.
1.17. Place the tube into a 37 °C water beaker with magnetic stirring at 500 rpm and incubate for 30 min  (vortex the tube every 15 min). 

1.21. Centrifuge the mixture at 2000 x g, 5 min  and discard the supernatant. 

1.20. Add 8 mL mL of washing buffer into the mixture and gently mix by pipetting for 3-5 times using a pre-wet serological pipette.

1.19. The enzymatic dissociation activity is stopped by a dilution technique. 
1.18. Refresh the enzymatic activity by repeating step   to   one more time.
 


1.26. Collect the flow-through cell suspension.
1.25. Wash the serological pipette and mesh filter with an additional 3 mL washing buffer.

1.24. Transfer the mixture by using the same pipette to the top of a mesh filter (    pore size).

1.23. Resuspend the cell pellet by adding 2 mL of washing buffer and mix vigorously with a pre-wet 10 mL serological pipet.

1.22. 1.2.1Repeat step 1.2.12-1.2.13 for two more times.
1.32. Assess the quality of the primary cells by determining the cell numbers and viability with the Trypan blue exclusion method and can confirm such counts with a 
 

 

  

1.31. Add 2 mL of culture media into the cell suspension and gently pipetting with a pre-wet P1000 pipette tip.

1.30. Carefully aspirate the supernatant by a pre-wet P1000 pipette tip (~300 µL of buffer can be left on the pellet). 

1.28. Wash the syringe and mesh strainer with an additional 5 mL washing buffer.


1.27. To make a single cell suspension solution, gently aspirate the flow-through cell suspension by using a 29 G syringe. Then, gently pass the suspension through a   mesh strainer by pressing against the mesh on a circular motion. 

1.29. Centrifuge the cell suspension at 1000 x g, 5 min and discard the supernatant. 

SECTION: Cell plating and culture
1.33. One day before cell plating, thaw a vial of   (BME) overnight in 4°C and use it for coating the growth surface area of a tissue culture flask T75 prior to cell culture.

SECTION: Cell plating and culture
1.38. Remove the mixture after incubation. The flask is ready for cell plating or can be kept in 4 °C   for plating on the following day.
SECTION: Cell plating and culture
1.37. Gently swirl the mixture to cover the entire growth surface area and incubate the flask at Room temperature for 60 min.
SECTION: Cell plating and culture
1.36. Pipetting solution up and down with a 5mL serological pipette by being careful not to create air bubbles and then transfer the mixture into a T75 tissue culture flask.
SECTION: Cell plating and culture
1.35. Place a vial of BME on ice during work to prevent untimely gelling. 
SECTION: Cell plating and culture
1.41. Observe the morphological heterogeneity (Figure 2) under a light microscope and replace the media every 2 days.
 
  

SECTION: Cell plating and culture
1.40. Incubate cells at 37 °C 5 % CO2.
SECTION: Cell plating and culture
1.39. For cell plating, pipette 1.0x105 cells in 10 mL of expansion media into a BME-coated T75 culture flask.
SECTION: Cell plating and culture
1.34. For T75 flask coating, pipette 10 µL of the BME into a 15 mL tube containing 5 mL of cold serum-free   media to make a 1:50 dilution.
SECTION: Cell passaging and epithelial enrichment and sorting
1.42. After the confluency of the monolayer cells reached 70%-80%, remove the old expansion media from a culture flask with a 10 mL serological pipette.
SECTION: Cell passaging and epithelial enrichment and sorting
1.52. To passage the cells or perform epithelial cell enrichment and/or sorting. Replace the old expansion medium with 10 mL of fresh EM or EEM at day 2 and replace the medium every 2880 min until cells reach the desired confluency. 
 
  

SECTION: Cell passaging and epithelial enrichment and sorting
1.45. Incubate with the enzyme solution at 37 °C  for 15 min. To enhance the cell dissociation process, remove the flask from the incubator to swirl or shake every 5 min.
SECTION: Cell passaging and epithelial enrichment and sorting
1.44. Discard the solution from the cells before pipetting 1 mL  of   to cover the monolayer cells.
SECTION: Cell passaging and epithelial enrichment and sorting
1.43. Transfer 10 mL of 1XPBS into the flask and incubate for 1 min.

SECTION: Cell passaging and epithelial enrichment and sorting
1.46. Observe the single cell dissociation phenomena under a light microscope.
SECTION: Cell passaging and epithelial enrichment and sorting
1.47. Stop the enzymatic reaction using a dilution technique: use a 10 mL serological pipette to transfer 9 mL of basal medium into a flask and resuspend the suspension by pipetting up and down for 3-5 times. 
SECTION: Cell passaging and epithelial enrichment and sorting
1.48. Transfer the suspension into a 15 mL conical tube and pellet the cells by centrifugation at 1000 x g, 5 min. 
SECTION: Cell passaging and epithelial enrichment and sorting
1.49. Discard the supernatant and resuspend the pellet by using 5 mL of expansion media.
SECTION: Cell passaging and epithelial enrichment and sorting
1.50. Assess the cell number and viability by using Trypan blue exclusion method and confirm the cell count with 
 

SECTION: Cell passaging and epithelial enrichment and sorting
1.51. Pipette 1.0x105 cells in 10 mL of expansion medium into a new BME-coated T75 tissue culture flask and incubate the cells at 37 °C 5 %CO2.
SECTION: Cell magnetic bioassembly and senescence induction
2.2. Then, resuspend the cell pellet with 1 mL of epithelial enrichment media (EEM).

2.3. 
Determine cell numbers and viability by Trypan blue exclusion method and confirm the count with a 
 
.
 


2.5. To fabricate 20 organoids, prepare 420 µL  of the cells at a density 1.0x106 cells/mL in a 50 mm dish by adjusting with EEM. 

2.4. To fabricate 20 organoids, prepare 420 µL μL of the cells at a density 1.0x106 cells/mL in a 50 mm dish by adjusting with EEM. 

2.5. Add 42 µL of the   magnetic nanoparticle solution or MNP into a cell suspension and gently mix with a pre-wet P1000 pipette tip.

2.6. Incubate the suspension for 120 min in a 37 °C  incubator, 5 % CO2. To ensure proper mixing during incubation, shake the tube on an orbital shaker at  .
2.6. Add 42 µL of the   magnetic nanoparticle solution or MNP into a cell suspension and gently mix with a pre-wet P1000 pipette tip.

2.7. 
Incubate the suspension for 120 min in a 37 °C incubator, 5 %CO2 with humidity. To ensure proper mixing during incubation, shake the tube on an orbital shaker at   

2.8. After incubation, centrifuge the cell-MNP solution at 800 rpm, 5 min and remove the supernatant by pipetting. 

2.9. Gently tap the cell pellet to resuspend the single cells and adjust the cell concentration to 1.33x105 cells/mL by adding 2730 µL of expansion media.

2.10. Pipette the mixture up and down with a P1000 pipette tip to ensure a single cell suspension.
2.11. Place the ultra-low attachment 96 well plate on top of a 96-well spheroid magnetic drive prior to bioassembly and bioprinting (Figure 4A).
2.12. Transfer 150 µL of cell suspension to each well of the plate. To prevent aggregation of the magnetized cells, gently hand shake the tube during pipetting.

2.13. Seal the border of the plate with 100 µL  of sterilized water to minimize the evaporation of media and incubate the plate in 37 °C 5 % CO2 with humidity for 180 min and observe cell morphology (Figure 4B).

2.14. Remove the magnetic drive from the bottom of the plate and incubate the plate further in 37 °C 5 % CO2 with humidity for 11520 min with medium replacement every 2880 min (Figure 4C). 

2.15. To induce cellular senescence, treat the organoids with 10 millimolar (mM)-100 millimolar (mM)  of  in EEM at day 8 for 1440 min .
  

3. Secretory LG/SG organoid validation
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.1. Use a magnetic holder to hold the organoid at the bottom of each well in the 96 well plate and remove all media from the organoids. Avoid sucking up the organoids and shear them through the P200 pipette tip 
 
 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.2. Gently wash the organoids with 200 µL  of 1X PBS and discard all solution. 

SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.3. Fix the organoids by adding 100 μL of 4% PFA and incubate at room temperature for 30 minutes with a 400 rpm. orbital swirling. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.4. Use a magnetic holder to hold the organoid in each well of 96 well plate and remove all solutions (4% PFA and later 1X PBS) from the organoids. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.5. Gently wash the organoids with 200 μL of 1X PBS and discard all solution for three times. 
 

SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.6. For immunofluorescent labeling, remove residual 1X PBS with a P200 pipette tip as much as possible. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.7. Permeabilize the organoids with 200 μL of 0.1% v/v Tween-20 for 20 minutes with 400 rpm orbital swirling and then remove all solution with a P200 pipette tip. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.8. Wash the organoids with 200μL of 1X PBS and remove the solution with a P200 pipette tip for at least three times. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.9. Add 200 μL of blocking buffer into the center of organoids and incubate for 6 hours at room temperature or 1-2 hours at room temperature followed by overnight at 4 °C in a humidified chamber with a 400 rpm orbital shaker. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.10. After incubation, remove the blocking buffer from organoids with a P200 pipette.
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.11. Add 100 μL solution of primary antibodies (against protein markers of acinar and ductal epithelial cells) into the organoids and incubate for at least 3 hours at room temperature or overnight at 4°C in a humidified chamber with a 400 rpm orbital sharker.
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.12. After incubation, remove the solution from the organoids with an P200 pipette tip and wash the excess of antibody solution with 200 μL of 0.5% v/v Triton X-100 in 1X PBS for 20 min with 400 rpm orbital shaker, at least three times. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.13. Add 100 μL of a solution with secondary antibodies into the organoids (specific to the host species of the previously used primary antibodies) and incubate at room temperature for 1 hour with a protection against photobleaching. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.14. After incubation, remove all solutions with secondary antibodies with a P200 pipette tip and rinse with 200 μL of a washing buffer solution containing 0.5% v/v Triton X-100 in 1X PBS for 20 minutes with 400 rpm orbital shaker, at least three times. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.15. Replace the solution with 100 μL of nuclear stain solution (10% v/v Hoechst 33342 in 1X PBS) and incubate at room temperature with 400 rpm orbital shaker for 1 hour. 
SECTION: Immunofluorescent staining of acinar and ductal epithelial compartments
3.16. Remove the nuclear stain solution and observe the labeled organoids under a fluorescence microscope before mounting them on a regular glass slide with a resin mounting media (Electron Microscopy Science, Hatfield, PA, USA).
SECTION: Primary cell extraction and isolation
1.9. Transfer 200 mg of gland tissue into a glass spot plate and add 500 µL of collection buffer to prevent tissue to dry out.

2.4. 
SECTION: Cell magnetic bioassembly and senescence induction
2.1. 2.1.1Before magnetization, dissociate the cells at the monolayer stage at the confluency of 70-80% by following the previous steps in section 1  . 

2.7. After incubation, centrifuge the cell-MNP solution at 800 x g, 5 min and remove the supernatant by pipetting. 

2.8. Gently tap the cell pellet to resuspend the single cells and adjust the cell concentration to 1.33x105 cells/mL by adding 2730 µL of expansion media.

2.9. Pipette the mixture up and down with a P1000 pipette tip to ensure a single cell suspension.
2.10. Place the ultra-low attachment 96 well plate on top of a 96-well spheroid magnetic drive prior to bioassembly and bioprinting (Figure 4A).
2.11. Transfer 150 µL of cell suspension to each well of the plate. To prevent aggregation of the magnetized cells, gently hand shake the tube during pipetting.

2.12. Seal the border of the plate with 100 µL of sterilized water to minimize the evaporation of media and incubate the plate in 37 °C , 5 %  CO2 with humidity for 180 min  and observe cell morphology (Figure 4B).

2.13. Remove the magnetic drive from the bottom of the plate and incubate the plate further in 37 °C , 5 % CO2 with humidity for 11520 min  with medium replacement every 2880 min  (Figure 4C).

2.14. To induce cellular senescence, treat the organoids with 10 millimolar (mM) -100 millimolar (mM) of    in EEM at day 8 for 1440 min.
  

3. Secretory LG/SG organoid validation
SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.1. Use a magnetic holder to hold the organoid at the bottom of each well in the 96 well plate and remove all media from the organoids. Avoid sucking up the organoids and shear them through the P200 pipette tip (always use normal uncut tip for this step).


SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.2. Gently wash the organoids with 200 µL of 1X PBS and discard all solution. 

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.3. Fix the organoids by adding 100 µL of 4 Mass / % volume    (PFA) and incubate at room temperature for 30 min with a   orbital swirling. 

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.4. Use a magnetic holder to hold the organoid in each well of 96 well plate and remove all solutions (4% PFA and later 1X PBS) from the organoids. 
SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.5. Gently wash the organoids with 200 µL of 1X PBS and discard all solution for three times. 
 


SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.6. For immunofluorescent labeling, remove residual 1X PBS with a P200 pipette tip as much as possible. 
SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.7. Permeabilize the organoids with 200 µL of 0.1 % (v/v) Triton X for 20 min with    orbital swirling and then remove all solution with a P200 pipette tip. 

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.8. Wash the organoids with 200 µL of 1X PBS and remove the solution with a P200 pipette tip for at least three times. 

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.9. Add 200 µL of blocking buffer into the center of organoids and incubate for 6 hours at room temperature or 60 min - 120 min  at Room temperature  followed by    at 4 °C  in a humidified chamber with a   orbital shaker. 

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.10. After incubation, remove the blocking buffer from organoids with a P200 pipette. 
SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.11. Add 100 µL solution of primary antibodies (against protein markers of acinar and ductal epithelial cells) into the organoids and incubate for at least 180 min at Room temperature or   4 °C in a humidified chamber with   orbital shaker.

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.12. After incubation, remove the solution from the organoids with a P200 pipette tip and wash the excess of antibody solution with 200 µL of 0.1 % (v/v) Tween-20 in 1X PBS for 20 min with   orbital shaker, at least three times. 

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.13. Add 100 µL  of a solution with secondary antibodies into the organoids (specific to the host species of the previously used primary antibodies) and incubate at room temperature for 60 min with a protection against photobleaching. 

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.14. After incubation, remove all solutions with secondary antibodies with a P200 pipette tip and rinse with 200 µL  of a washing buffer solution containing 0.5 % (v/v) Tween-20 in 1X PBS for 20 min with    orbital shaker, at least three times. 

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.15. Replace the solution with 100 µL of nuclear stain solution (10 % (v/v) Hoechst 33342 in 1X PBS) and incubate at Room temperaturewith   orbital shaker for 60 min.

SECTION: 3.1	Immunofluorescent staining of acinar and ductal epithelial compartments
3.16. Remove the nuclear stain solution and observe the labeled organoids under a fluorescence microscope before mounting them on a regular glass slide with a  resin mounting media. 

Antibodies used 
 

Solutions/media used