Sep 10, 2024

Public workspaceMulticellular Circulating Co-Culture

DOI
dx.doi.org/10.17504/protocols.io.ewov19b47lr2/v1
  • Bianca Cruz Pachane1,
  • Pedro Henrique Teixeira Bottaro1,
  • Wanessa Fernanda Altei2,
  • Heloisa Sobreiro Selistre de Araujo1
  • 1Universidade Federal de São Carlos - UFSCar, São Carlos, SP, Brazil;
  • 2Barretos Cancer Hospital, Barretos, SP, Brazil
  • Bianca Cruz Pachane: Full optimization and experimentation;
  • Pedro Henrique Teixeira Bottaro: Optimization of THP-1 culture
  • Wanessa Fernanda Altei: Initial development of method
  • Heloisa Sobreiro Selistre de Araujo: Financial support and System acquisition
Bianca Cruz Pachane
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DOI: dx.doi.org/10.17504/protocols.io.ewov19b47lr2/v1
Protocol Citation: Bianca Cruz Pachane, Pedro Henrique Teixeira Bottaro, Wanessa Fernanda Altei, Heloisa Sobreiro Selistre de Araujo 2024. Multicellular Circulating Co-Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19b47lr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 10, 2024
Last Modified: September 10, 2024
Protocol Integer ID: 107221
Funders Acknowledgement:
São Paulo Research Foundation
Grant ID: 2021/01983-4
São Paulo Research Foundation
Grant ID: 2019/11437-7
São Paulo Research Foundation
Grant ID: 2022/12307-2
Disclaimer
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Abstract
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.
Image Attribution
The diagram was created using Adobe Photoshop. Original photograph by Bianca Pachane.
Materials
Materials:
  1. Round glass coverslips 13mm ø
  2. 24-well clear plates with flat bottom
  3. Histological slides, Exacta.
  4. Sterile forceps
  5. Petri dishes
  6. 0.22 pore syringe filters

Reagents and Solutions:
  1. ReagentParafilm™ M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410
  2. ReagentPoly-l-lysine, 0.1% (wt/vol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8920
  3. ReagentGlutaraldehyde solution (50% in solution)Merck MilliporeSigma (Sigma-Aldrich)Catalog #G6403
  4. ReagentGelatin From Pig Skin, Fluorescein ConjugateInvitrogen - Thermo FisherCatalog #G13187
  5. ReagentCorning® Matrigel®CorningCatalog #354277
  6. ReagentFibronectinGibco - Thermo FisherCatalog #33016-015
  7. Reagent1X PBS (Phosphate-buffered saline )
  8. ReagentOptiMEM™ I Reduced Serum MediaGibco, ThermoFisherCatalog #31985070
  9. ReagentTrypan Blue Solution 0.4% Sterile-filtered Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8154
  10. ReagentCellTracker™ Red CMTPX DyeThermo FisherCatalog #C34552 - reconstituted in DMSO
  11. ReagentParaformaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6148 - PFA 4% in deionized water, pH 7.6, sterile
  12. ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML - Triton X-100 0.1% (v/v) in deionized water
  13. ReagentBovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7030 - 1% BSA-PBS
  14. ReagentAnti-VEGF Receptor 2 antibodyAbcamCatalog #ab39256 - 1:50 dilution in 1% BSA-PBS
  15. ReagentBD Transduction Laboratories™ Purified Mouse Anti-β-CateninBD BiosciencesCatalog #610154 - 1:50 dilution in 1% BSA-PBS
  16. ReagentAnti-Collagen II antibodyAbcamCatalog #ab85266 - 1:50 dilution in 1% BSA-PBS
  17. ReagentAnti-Collagen III antibody [1E7-D7/Col3]AbcamCatalog #ab23445 - 1:50 dilution in 1% BSA-PBS
  18. ReagentGoat Anti-Mouse IgG H&L (FITC)AbcamCatalog #ab6785 - 1:1,000 dilution in 1% BSA-PBS
  19. ReagentGoat Anti-Rabbit IgG H&L (Alexa Fluor® 568)AbcamCatalog #ab175696 - 1:1,000 dilution in 1% BSA-PBS
  20. Phalloidin + DAPI (1 µl ReagentPhalloidin-iFluor 647 ReagentAbcamCatalog #ab176759 + 0.76 µl Reagent4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)Thermo Fisher ScientificCatalog #D1306 in 5 ml PBS)
  21. ReagentFluoromountMerck MilliporeSigma (Sigma-Aldrich)Catalog #F4680

Cell lines and growth media:
  1. MDA-MB-231 (ATCC™ CRM-HTB-26®) - Leibovitz L-15 10% FBS
  2. HDFa (ATCC™ PCS-201-012®) - DMEM 10% FBS 1% pen/strep
  3. HUVEC (ATCC™ CRL-1730®) - DMEM 10% FBS 1% pen/strep
  4. THP-1 (ATCC™ TIB-202®) - RPMI 1640 10% FBS 1% pen/strep

Equipments:
  1. Biological cabinet
  2. Cell incubator (37 ºC, 5% CO2)
  3. QV500 System, Kirstall
  4. Cell counter - TC20 Cell Counter, Bio-Rad - Catalog #1450011
  5. Epifluorescence microscope - ImageXpress Micro XLS, Molecular Devices - Catalog #500496


Protocol materials
ReagentParafilm™ M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410
In Materials and 4 steps
ReagentCellTracker™ Red CMTPX DyeThermo FisherCatalog #C34552
Materials, Step 22
ReagentAnti-Collagen III antibody [1E7-D7/Col3]AbcamCatalog #ab23445
Materials, Step 46
ReagentCorning® Matrigel®CorningCatalog #354277
Materials
ReagentFibronectinGibco - Thermo FisherCatalog #33016-015
Materials
ReagentPhalloidin-iFluor 647 ReagentAbcamCatalog #ab176759
Materials, Step 56
ReagentTrypan Blue Solution 0.4% Sterile-filtered Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8154
In Materials and 3 steps
ReagentFluoromountMerck MilliporeSigma (Sigma-Aldrich)Catalog #F4680
Materials, Step 58
ReagentAnti-VEGF Receptor 2 antibodyAbcamCatalog #ab39256
Materials, Step 46
ReagentGoat Anti-Rabbit IgG H&L (Alexa Fluor® 568)AbcamCatalog #ab175696
Materials, Step 51
ReagentOptiMEM™ I Reduced Serum MediaGibco, ThermoFisherCatalog #31985070
In Materials and 7 steps
ReagentPoly-l-lysine, 0.1% (wt/vol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8920
Materials, Step 3
ReagentBD Transduction Laboratories™ Purified Mouse Anti-β-CateninBD BiosciencesCatalog #610154
Materials, Step 46
ReagentGelatin From Pig Skin, Fluorescein ConjugateInvitrogen - Thermo FisherCatalog #G13187
Materials
Reagent4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)Thermo Fisher ScientificCatalog #D1306
Materials, Step 56
ReagentGlutaraldehyde solution (50% in solution)Merck MilliporeSigma (Sigma-Aldrich)Catalog #G6403
Materials, Step 2
ReagentParaformaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6148
Materials
ReagentGoat Anti-Mouse IgG H&L (FITC)AbcamCatalog #ab6785
Materials, Step 51
ReagentBovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7030
Materials
ReagentAnti-Collagen II antibodyAbcamCatalog #ab85266
Materials, Step 46
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Materials
Safety warnings
Light-sensitive assay. Work under sterile conditions.
Before start
Fluorescent gelatin preparation: Under sterile conditions, solubilize the fluorescent gelatin stock at Temperature37 °C with warmed PBS following the manufacturer's instructions for a concentration of Concentration5 mg/mL . Aliquot in microtubes and maintain at Temperature-20 °C until time of use.
Before use, thaw gelatin at Temperature37 °C for Duration00:30:00 . Dilute stock to a Concentration0.2 mg/mL working solution with warmed PBS and maintain at Temperature37 °C until use.

Matrigel preparation: Thaw Matrigel vial at Temperature4 °C and maintain TemperatureOn 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.
Preparation of matrix-coated coverslips
Preparation of matrix-coated coverslips
20m
20m
Clean round glass coverslips (13 mm ø) with 70% ethanol wipes before use. Maintain slips in a clean container.
Prepare a 0.5% solution of glutaraldehyde in H2O and maintain it at Temperature4 °C protected from light.
ReagentGlutaraldehyde solution (50% in solution)Merck MilliporeSigma (Sigma-Aldrich)Catalog #G6403
Under sterile conditions, prepare a surface with a piece of Parafilm fixed in place. Add Amount20 µL droplets of poly-L-lysine interspaced in the Parafilm.
ReagentPoly-l-lysine, 0.1% (wt/vol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8920
ReagentParafilm™ M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410
Drop coverslips atop the droplets and incubate at TemperatureRoom temperature for Duration00:20:00 minimum.
20m
Incubation
Using forceps, transfer the coverslips to a 24-well plate with the coating facing upwards.
Wash coverslips twice withAmount500 µL PBS.
Reagent1X PBS (Phosphate-buffered saline )
Cross-link coating with Amount500 µL of cold Concentration0.5 % (v/v) glutaraldehyde for Duration00:15:00 at TemperatureRoom temperature
15m
Incubation
Prepare a Petri dish with the bottom covered in Parafilm.
ReagentParafilm™ M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410
Apply spaced Amount20 µL droplets of the matrices to the Parafilm-covered surface:
  • Fluorescent gelatin: Concentration0.2 mg/mL in PBS, kept at Temperature37 °C until time of use;
  • Matrigel solution: Concentration50 % (v/v) in PBS, kept TemperatureOn ice until time of use;
  • Fibronectin solution: Concentration1 mg/mL in PBS, kept TemperatureOn ice until time of use;
Critical
Remove the coverslips from the 24-well plate and drop them atop the droplets, with the coating facing down. Incubate at Temperature4 °C DurationOvernight , protected from light.
Incubation
Overnight
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.
Wash coverslips twice withAmount500 µL PBS.
Reagent1X PBS (Phosphate-buffered saline )
Slips can be stored at 4 ºC for up to a week, wrapped in aluminium foil.
Optional
Pause
Pre-condition matrix coating with Amount500 µL of culture media for Duration00:30:00 at Temperature37 °C :
  • Fluorescent gelatin: Leibovitz L-15 10% FBS
  • Matrigel solution: DMEM 10% FBS 1% pen/strep
  • Fibronectin solution: DMEM 10% FBS 1% pen/strep
30m
Incubation
Cell seeding
Cell seeding
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
ReagentTrypan Blue Solution 0.4% Sterile-filtered Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8154
Remove the pre-conditioning medium from the 24-well plate.
Seed cells in the 24-wells following the table below:

ABCD
Coating:Cell Type:Density:Culture Media:
Fluorescent gelatinMDA-MB-23150,000 cells/ml1 ml Leibovitz L-15 10% FBS
MatrigelHUVEC50,000 cells/ml1 ml DMEM 10% FBS 1% pen/strep
FibronectinHDFa20,000 cells/ml1 ml DMEM 10% FBS 1% pen/strep

Incubate cells at Temperature37 °C 5% CO2 DurationOvernight for adhesion.
ps: seal the MDA-MB-231 plate with parafilm to protect from CO2 exposure.
Incubation
Critical
THP-1 staining and seeding
THP-1 staining and seeding
5m
5m
The day after cell seeding:
Transfer THP-1 suspension to a conical tube and centrifuge at Centrifigation1200 rpm, 00:05:00 . Discard supernatant.
5m
Resuspend cell pellet in OptiMEM 1% pen/strep and count cells using the trypan blue exclusion method.
ReagentOptiMEM™ I Reduced Serum MediaGibco, ThermoFisherCatalog #31985070
ReagentTrypan Blue Solution 0.4% Sterile-filtered Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8154
Dilute cell suspension to a 1x106 cells/ml density in OptiMEM 1% pen/strep.
ReagentOptiMEM™ I Reduced Serum MediaGibco, ThermoFisherCatalog #31985070
Add Amount1 µL of CellTracker™ CMPTX per ml of cell suspension for a Concentration5 micromolar (µM) final concentration. Pipette well to mix.
ReagentCellTracker™ Red CMTPX DyeThermo FisherCatalog #C34552
Incubate the cell suspension atTemperature37 °C % CO2 for Duration00:30:00 , protected from light.

30m
Dilute cell suspensions with Amount4 mL OptiMEM and centrifuge at Centrifigation1200 rpm, 00:05:00
ReagentOptiMEM™ I Reduced Serum MediaGibco, ThermoFisherCatalog #31985070
5m
Resuspend cell pellets in Amount1 mL OptiMEM and recount cells using the trypan blue exclusion method.
ReagentOptiMEM™ I Reduced Serum MediaGibco, ThermoFisherCatalog #31985070 ReagentTrypan Blue Solution 0.4% Sterile-filtered Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8154
In a 15-ml conical tube, prepare a 1x105 cell/ml THP-1 suspension in Amount15 mL OptiMEM 1% pen/strep . Maintain protected from light.
ReagentOptiMEM™ I Reduced Serum MediaGibco, ThermoFisherCatalog #31985070
QV500 assembly and preparation
QV500 assembly and preparation
10m
10m
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.
Assemble the system under the biological hood with autoclaved parts:
Add Amount15 mL sterile PBS to the reservoir and close the lid.
Reagent1X PBS (Phosphate-buffered saline )
Attach the 0.22 µm pore syringe filter to the air outlet (blue piece).
Add Amount1 mL sterile PBS to each chamber and close the lids.
Reagent1X PBS (Phosphate-buffered saline )
Attach the 1.6 mm pump tubing to the peristaltic pump.
Connect the inlet and outlet tubings with the chambers to form a close circuit.
Circulate the PBS for Duration00:10:00 at max flow rate.

10m
Incubation
Drain the system and disassemble the parts.
Repeat steps Go togo to step #28.1 until step #29 with 15 ml OptiMEM to condition the system.
ReagentOptiMEM™ I Reduced Serum MediaGibco, ThermoFisherCatalog #31985070
Multicellular Circulating Co-Culture
Multicellular Circulating Co-Culture
1d
1d
Remove the growth media from cells seeded in coverslips (Go togo to step #18 ).

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.
Critical
Add Amount1 mL 1 ml OptiMEM 1% pen/strep to each chamber and close the lids, connecting the tubings following the order described above.
ReagentOptiMEM™ I Reduced Serum MediaGibco, ThermoFisherCatalog #31985070

Add the Amount15 mL THP-1 cell suspension in OptiMEM 1% pen/strep to the reservoir.

Add EVh (109 particles/ml) or the equivalent treatment volume in PBS to the reservoir and close the lid.
Connect the tubings to close the system. Circulate the media at a 50 µl/s flow rate and incubate the whole system for Duration24:00:00 at Temperature37 °C 5% CO2.
1d
Incubation
Overnight
The next day, disconnect the system and carefully drain the compartments. Collect the conditioned media in a conical tube and spin at Centrifigation1200 rpm, 4°C, 00:10:00 . Transfer the supernatant to fresh tubes and freeze at Temperature-80 °C for further testing.

10m
Centrifigation
Transfer the coverslips to a fresh 24-well plate for fixing and staining (Go togo to step #40 )

Wash and decontaminate the QV500 parts for further use.
Cell Fixing
Cell Fixing
Fix cells in coverslips with Amount500 µL warmed 4% PFA atTemperatureRoom temperature for Duration00:10:00

10m
Incubation
Wash coverslips twice withAmount500 µL PBS.
Reagent1X PBS (Phosphate-buffered saline )
Permeabilize cells with Amount500 µL 0.1% Triton X-100 at TemperatureRoom temperature for Duration00:05:00

5m
Incubation
Wash coverslips twice withAmount500 µL PBS.
Reagent1X PBS (Phosphate-buffered saline )
For MDA-MB-231 coverslips only: skip the immunofluorescence protocol and Go togo to step #56
Immunofluorescence of HUVEC and HDFa Coverslips
Immunofluorescence of HUVEC and HDFa Coverslips
Block non-specific bindings with Amount500 µL 1% BSA-PBS for Duration01:00:00 at Temperature4 °C
1h
Prepare the primary antibody dilutions:

For HUVECs:
  1. ReagentAnti-VEGF Receptor 2 antibodyAbcamCatalog #ab39256 - 1:50 dilution in 1% BSA-PBS
  2. ReagentBD Transduction Laboratories™ Purified Mouse Anti-β-CateninBD BiosciencesCatalog #610154 - 1:50 dilution in 1% BSA-PBS

For HDFa:
  1. ReagentAnti-Collagen II antibodyAbcamCatalog #ab85266 - 1:50 dilution in 1% BSA-PBS
  2. ReagentAnti-Collagen III antibody [1E7-D7/Col3]AbcamCatalog #ab23445 - 1:50 dilution in 1% BSA-PBS
Pipetting
Place Amount20 µL of spaced droplets of the antibodies in a Petri dish covered with Parafilm (as seen on Go togo to step #8 ).
ReagentParafilm™ M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410
Remove the coverslips from the 24-well plate and drop them atop the droplets, with the cells facing down. Incubate at Temperature4 °C for Duration18:00:00 , protected from light.
18h
Incubation
Overnight
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.
Wash coverslips twice withAmount500 µL PBS.
Reagent1X PBS (Phosphate-buffered saline )
Prepare the secondary antibody dilutions:

  1. ReagentGoat Anti-Mouse IgG H&L (FITC)AbcamCatalog #ab6785 - 1:1,000 dilution in 1% BSA-PBS
  2. ReagentGoat Anti-Rabbit IgG H&L (Alexa Fluor® 568)AbcamCatalog #ab175696 - 1:1,000 dilution in 1% BSA-PBS
Place Amount20 µL of spaced droplets of the antibodies in a Petri dish covered with Parafilm (as seen on Go togo to step #8 ).
ReagentParafilm™ M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410
Remove the coverslips from the 24-well plate and drop them atop the droplets, with the cells facing down. Incubate at TemperatureRoom temperature for Duration01:00:00 , protected from light.
1h
Incubation
Remove the slips from the Petri dish using a forceps and transfer them, with the coating facing up, to a fresh 24-well plate.
Wash coverslips twice withAmount500 µL PBS.
Reagent1X PBS (Phosphate-buffered saline )
Cell Counterstaining and Slide Assembly
Cell Counterstaining and Slide Assembly
20m
20m
Add Amount500 µL of DAPI + Phalloidin-647 mixture to each well and incubate for Duration00:20:00 .

> 1 µl ReagentPhalloidin-iFluor 647 ReagentAbcamCatalog #ab176759 + 0.76 µl Reagent4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)Thermo Fisher ScientificCatalog #D1306 in 5 ml PBS
20m
Incubation
Wash coverslips twice withAmount500 µL PBS.
Reagent1X PBS (Phosphate-buffered saline )
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 Duration04:00:00 .
ReagentFluoromountMerck MilliporeSigma (Sigma-Aldrich)Catalog #F4680

4h
Incubation
Once dry, seal coverslips using clear nail polish and store atTemperature4 °C until time of analysis.

Pause
Cell Imaging by Epifluorescence HTS
Cell Imaging by Epifluorescence HTS
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).
Set laser intensity to a minimum of 10 ms and increase gradatively if necessary.
Check the wells using the 4X objective.
Change into the 20x objective and adjust the laser focus. Select 9 sites per well minimally.
Acquire the plate. Export metadata for analysis.
Repeat Go togo to step #63 and step #64 with the 40x objective.

For representative images, use the 40x objective and adjust the laser focus. Select the sites of interest and acquire. Export image channels and combinations.
Imaging analysis
Imaging analysis
Gelatin degradation quantification, cell morphology analysis, quantification of immunofluorescence probing, and assembly of representative images were performed using FIJI.
Software
ImageJ/Fiji
NAME
Windows 7
OS
National Institutes of Health
DEVELOPER
http://wsr.imagej.net/distros/win/ij152-win-java8.zip
SOURCE LINK
Please refer to the following protocol for the complete pipeline of analysis.
Protocol
Cell Invasion in Direct Co-Culture
NAME
Cell Invasion in Direct Co-Culture
CREATED BY
Bianca Cruz Pachane

Protocol references
PACHANE, Bianca Cruz et al. Small Extracellular Vesicles from Hypoxic Triple-Negative Breast Cancer Cells Induce Oxygen-Dependent Cell Invasion. International Journal of Molecular Sciences, [s. l.], v. 23, n. 20, p. 12646, 2022.
EVEN-RAM, Sharona; ARTYM, Vira. Extracellular Matrix Protocols: Second Edition. [S. l.]: Humana Press, 2009.