TITLE: Multicellular Circulating Co-Culture
AUTHORS: Bianca Cruz Pachane, Pedro Henrique Teixeira Bottaro, Wanessa Fernanda Altei, Heloisa Sobreiro Selistre de Araujo

[DESCRIPTION]
A novel method to study the tumor microenvironment (TME) in vitro, using the quasi-vivo technology from Kirstall to survey the individual responses in cell types common to the TME. We have developed a strategy that allows a tumoral cell (MDA-MB-231) seeded in gelatin coating, an endothelial cell (HUVEC) seeded in Matrigel coating, and a dermal fibroblast (HDFa) seeded in fibronectin to be cultured in tandem, alongside suspension monocytes (THP-1). The goal was to investigate how cells would behave in this setting and evaluate the role of hypoxic tumoral extracellular vesicles in the development of the TME.

[BEFORE_START]
Fluorescent gelatin preparation: Under sterile conditions, solubilize the fluorescent gelatin stock at 37 °Cwith warmed PBS following the manufacturer's instructions for a concentration of 5 mg/mLAliquot in microtubes and maintain at -20 °Cuntil time of use. 
Before use, thaw gelatin at 37 °Cfor 30 minDilute stock to a 0.2 mg/mLworking solution with warmed PBS and maintain at 37 °Cuntil use. 

Matrigel preparation: Thaw Matrigel vial at 4 °C  and maintain on ice until use. Dilute a 1:1, v/v aliquot of stock Matrigel in cold sterile PBS for use.

Cell culture: Maintain cells in culture during at least two passages after thawing.

QV500: Autoclave all parts before use.

[STEPS]
SECTION: Preparation of matrix-coated coverslips
1. Clean round glass coverslips (13 mm ø) with 70% ethanol wipes before use. Maintain slips in a clean container.
SECTION: Preparation of matrix-coated coverslips
2. Prepare a 0.5% solution of glutaraldehyde in H2O and maintain it at 4 °C protected from light.
  
SECTION: Preparation of matrix-coated coverslips
3. Under sterile conditions, prepare a surface with a piece of Parafilm fixed in place. Add 20 µL droplets of poly-L-lysine interspaced in the Parafilm.
  
  
SECTION: Preparation of matrix-coated coverslips
4. Drop coverslips atop the droplets and incubate at Room temperaturefor 20 min
SECTION: Preparation of matrix-coated coverslips
5. Using forceps, transfer the coverslips to a 24-well plate with the coating facing upwards. 
SECTION: Preparation of matrix-coated coverslips
6.  Wash coverslips twice with500 µL 
  
SECTION: Preparation of matrix-coated coverslips
7. Cross-link coating with 500 µLof cold 0.5 % (v/v) for 15 min  at Room temperature
SECTION: Preparation of matrix-coated coverslips
9. Apply spaced 20 µLdroplets of the matrices to the Parafilm-covered surface:
Fluorescent gelatin: 0.2 mg/mL kept at 37 °C until time of use;
Matrigel solution: 50 % (v/v)kept on ice until time of use;
Fibronectin solution:  1 mg/mL kept on ice until time of use;
SECTION: Preparation of matrix-coated coverslips
8. Prepare a Petri dish with the bottom covered in Parafilm.
  
SECTION: Preparation of matrix-coated coverslips
10. Remove the coverslips from the 24-well plate and drop them atop the droplets, with the coating facing down. Incubate at 4 °C protected from light.
SECTION: Preparation of matrix-coated coverslips
11. The next day, remove the slips from the Petri dish using a forceps and transfer them, with the coating facing up, to a fresh 24-well plate.
SECTION: Preparation of matrix-coated coverslips
13. Slips can be stored at 4 ºC for up to a week, wrapped in aluminium foil.
SECTION: Preparation of matrix-coated coverslips
14. Pre-condition matrix coating with 500 µLfor 30 minat 37 °C
Fluorescent gelatin: Leibovitz L-15 10% FBS
Matrigel solution: DMEM 10% FBS 1% pen/strep
Fibronectin solution: DMEM 10% FBS 1% pen/strep
SECTION: Preparation of matrix-coated coverslips
12.  Wash coverslips twice with500 µL 
  
SECTION: Cell seeding
15. Subculture cells as usual. Resuspend cell pellets in growth media and count cells using the trypan blue exclusion method.
MDA-MB-231 - Leibovitz L-15 10% FBS
HUVEC - DMEM 10% FBS 1% pen/strep
HDFa - DMEM 10% FBS 1% pen/strep 
  
SECTION: Cell seeding
16. Remove the pre-conditioning medium from the 24-well plate.
SECTION: Cell seeding
17. Seed cells in the 24-wells following the table below:

 

SECTION: Cell seeding
18. Incubate cells at 37 °C for adhesion.
ps: seal the MDA-MB-231 plate with parafilm to protect from CO2 exposure.
SECTION: THP-1 staining and seeding
19. The day after cell seeding:
Transfer THP-1 suspension to a conical tube and centrifuge at 1200 rpm, 5 min . Discard supernatant.
SECTION: THP-1 staining and seeding
20. Resuspend cell pellet in OptiMEM 1% pen/strep and count cells using the trypan blue exclusion method.
  
  
SECTION: THP-1 staining and seeding
21. Dilute cell suspension to a 1x106 cells/ml density in OptiMEM 1% pen/strep.
  
SECTION: THP-1 staining and seeding
22. Add 1 µL per ml of cell suspension for a 5 micromolar (µM) final concentration. Pipette well to mix.
  
SECTION: THP-1 staining and seeding
23. Incubate the cell suspension at37 °C  for 30 minprotected from light. 

SECTION: THP-1 staining and seeding
24. Dilute cell suspensions with 4 mLand centrifuge at 1200 rpm, 5 min
  
SECTION: THP-1 staining and seeding
25. Resuspend cell pellets in 1 mLand recount cells using the trypan blue exclusion method.
    
SECTION: THP-1 staining and seeding
26. In a 15-ml conical tube, prepare a 1x105 cell/ml THP-1 suspension in 15 mL. Maintain protected from light.
  
SECTION: QV500 assembly and preparation
27. QV500 consists of:
(1) Peristaltic pump
(3) Culture chambers with connecting tubings
(1) 15-ml reservoir with 1 inlet, 1 outlet and 1 air filter piece.
(1) 1.6 mm pump tubing
(2) plastic chamber supports

Check the experimental diagram for better understanding.
SECTION: QV500 assembly and preparation
28. Assemble the system under the biological hood with autoclaved parts:
SECTION: QV500 assembly and preparation
28.1. Add 15 mL to the reservoir and close the lid.
  
SECTION: QV500 assembly and preparation
28.2. Attach the 0.22 µm pore syringe filter to the air outlet (blue piece).
SECTION: QV500 assembly and preparation
28.3. Add 1 mL to each chamber and close the lids.
  
SECTION: QV500 assembly and preparation
28.4. Attach the 1.6 mm pump tubing to the peristaltic pump.
SECTION: QV500 assembly and preparation
28.5. Connect the inlet and outlet tubings with the chambers to form a close circuit.
SECTION: QV500 assembly and preparation
28.6. Circulate the PBS for 10 min at max flow rate.

SECTION: QV500 assembly and preparation
30. Repeat steps   with 15 ml OptiMEM to condition the system.
  
SECTION: QV500 assembly and preparation
29. Drain the system and disassemble the parts.
SECTION: Multicellular Circulating Co-Culture
31. Remove the growth media from cells seeded in coverslips (  ).

SECTION: Multicellular Circulating Co-Culture
32. Assemble the MDA-gelatin coverslip in one QV500 culture chamber; the HUVEC-Matrigel coverslip in the second culture chamber; and the HDFa-FN coverslip in the last culture chamber.
SECTION: Multicellular Circulating Co-Culture
33. Add 1 mL to each chamber and close the lids, connecting the tubings following the order described above.
   

SECTION: Multicellular Circulating Co-Culture
34. Add the 15 mL to the reservoir.

SECTION: Multicellular Circulating Co-Culture
36. Connect the tubings to close the system. Circulate the media at a 50 µl/s flow rate and incubate the whole system for 1440 min at 37 °C  
SECTION: Multicellular Circulating Co-Culture
35. Add EVh (109 particles/ml) or the equivalent treatment volume in PBS to the reservoir and close the lid.
SECTION: Multicellular Circulating Co-Culture
37. The next day, disconnect the system and carefully drain the compartments. Collect the conditioned media in a conical tube and spin at 1200 rpm, 10 min, 4 °C . Transfer the supernatant to fresh tubes and freeze at -80 °C for further testing.

SECTION: Multicellular Circulating Co-Culture
38. Transfer the coverslips to a fresh 24-well plate for fixing and staining (  )

SECTION: Multicellular Circulating Co-Culture
39. Wash and decontaminate the QV500 parts for further use.
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
45. Block non-specific bindings with 500 µL for 60 min at 4 °C 
SECTION: Cell Fixing
40. Fix cells in coverslips with 500 µLatRoom temperaturefor 10 min


SECTION: Cell Fixing
42. Permeabilize cells with 500 µL at Room temperature for 5 min 

SECTION: Cell Fixing
41.  Wash coverslips twice with500 µL 
  
SECTION: Cell Fixing
43.  Wash coverslips twice with500 µL 
  
SECTION: Cell Fixing
44. For MDA-MB-231 coverslips only: skip the immunofluorescence protocol and   
SECTION: Cell Counterstaining and Slide Assembly
56. Add 500 µL  DAPI + Phalloidin-647 mixture to each well and incubate for 20 min .

> 1 µl    + 0.76 µl    in 5 ml PBS
SECTION: Cell Counterstaining and Slide Assembly
58. Remove the coverslips from the 24-well plate and assemble them in histological slides with mounting media. Allow the media to dry for at least 240 min.
  

SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
46. Prepare the primary antibody dilutions:

For HUVECs:
   - 1:50 dilution in 1% BSA-PBS
   - 1:50 dilution in 1% BSA-PBS

For HDFa:
   - 1:50 dilution in 1% BSA-PBS
   - 1:50 dilution in 1% BSA-PBS
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
47. Place 20 µLof the antibodies in a Petri dish covered with Parafilm (as seen on  ).
  
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
51. Prepare the secondary antibody dilutions:

   - 1:1,000 dilution in 1% BSA-PBS
  - 1:1,000 dilution in 1% BSA-PBS
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
48. Remove the coverslips from the 24-well plate and drop them atop the droplets, with the cells facing down. Incubate at 4 °C1080 minprotected from light.
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
49. The next day, remove the slips from the Petri dish using a forceps and transfer them, with the coating facing up, to a fresh 24-well plate.
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
50.  Wash coverslips twice with500 µL 
  
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
52. Place 20 µLof the antibodies in a Petri dish covered with Parafilm (as seen on  ).
  
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
53. Remove the coverslips from the 24-well plate and drop them atop the droplets, with the cells facing down. Incubate at 4 Room temperature60 minprotected from light.
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
54. Remove the slips from the Petri dish using a forceps and transfer them, with the coating facing up, to a fresh 24-well plate.
SECTION: Immunofluorescence of HUVEC and HDFa Coverslips
55.  Wash coverslips twice with500 µL 
  
SECTION: Cell Counterstaining and Slide Assembly
57.  Wash coverslips twice with500 µL 
  
SECTION: Cell Counterstaining and Slide Assembly
59. Once dry, seal coverslips using clear nail polish and store at4 °Cuntil time of analysis.  

SECTION: Cell Imaging by Epifluorescence HTS
66. For representative images, use the 40x objective and adjust the laser focus. 
Select the sites of interest and acquire. 
Export image channels and combinations.
SECTION: Cell Imaging by Epifluorescence HTS
60. Using the microscope ImageXpress Micro XLS+ (Molecular Devices), check the template for the Corning 3603 plate and the filters for DAPI (nuclei), FITC (gelatin), TxRed (CMPTX) and Cy5 (phalloidin-647).
SECTION: Cell Imaging by Epifluorescence HTS
61. Set laser intensity to a minimum of 10 ms and increase gradatively if necessary.
SECTION: Cell Imaging by Epifluorescence HTS
62. Check the wells using the 4X objective.
SECTION: Cell Imaging by Epifluorescence HTS
63. Change into the 20x objective and adjust the laser focus. Select 9 sites per well minimally.
SECTION: Cell Imaging by Epifluorescence HTS
64. Acquire the plate. Export metadata for analysis.
SECTION: Cell Imaging by Epifluorescence HTS
65. Repeat   with the 40x objective.

SECTION: Imaging analysis
67. Gelatin degradation quantification, cell morphology analysis, quantification of immunofluorescence probing, and assembly of representative images were performed using FIJI.
 
Please refer to the following protocol for the complete pipeline of analysis.