3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Summary

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Abstract

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

Introduction

In physiological environment, cells are embedded in an extracellular matrix (ECM) and exposed to a plethora of biomolecules. Interactions between cells and the surrounding microenvironment regulate intracellular processes controlling diverse phenotypes, including migration, growth, differentiation and survival^1,2. Much has been learned about cellular behaviors in a conventional 2D cell culture. However, with the advent of intravital imaging and experimentation with cells embedded in 3D hydrogels, important differences in cell behaviors have been recognized in the simplified 2D in vitro cultures vs. 3D tissue-like environments. While cells interact with ECM fibers and sense their mechanical properties within the 3D matrix, the material stiffness of the gel is not a fully independent variable in a 2D in vitro system. The dimensionality alters focal adhesion formation, resulting in different cell morphology and behavior. Furthermore, cells on a 2D surface are exposed to fewer signaling cues than cells open to all directions in 3D.

These limitations have increased the interests for 3D systems that represent the spatial and chemical complexity of living tissues and better predict in vivo tissue functions. They have been developed in many forms from organoids as self-assembling cellular microstructures to cells randomly interspersed in ECM^3,4. Recent advances in microfabrication technologies have facilitated the advent of various types of 3D culture systems^5,6,7,8,9 for studying phenotypic changes and cellular responses to soluble signals; however, technological barriers limit the widespread use in research laboratories. In many cases, the fabrication processes require photolithography techniques and background knowledges for soft lithography. Moreover, various factors must be controlled to successfully build a device and to achieve an optimal function of the device over a long period of time.

Our method describes how to construct a 3D PDMS device for incorporating cells and multicellular organoids into a 3D microenvironment with defined chemoattractant gradients and then analyze epithelial responses to EGF¹⁰. Our data reveal that the capacity of organoids to respond to shallow EGF gradients arises from intercellular chemical coupling through gap junctions. It suggests the potential of organoids for more precise detection of weak and noisy spatially graded inputs. The fabrication process does not require a cleanroom facility nor photolithography techniques. However, the 3D PDMS device includes necessary factors of 3D physiological environment. This method will be helpful to study 3D cellular behaviors and it has great research potential with different cell types, chemoattractants, and ECM combinations.

Protocol

All animal work was conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee, Johns Hopkins University, School of Medicine.

1. Fabrication of the mesofluidic device

1. Design the mask of the mold for PDMS device using a 3D CAD software.
2. Print the mold using stereolithography equipment with a thermal resistant resin.
   NOTE: The procedures described here were carried out by a commercial 3D printing service.
3. Mix thoroughly a PDMS monomer solution with the curing agent in a 10:1 ratio. 3 mL of PDMS mixture was required for fabrication of the device. The total volume depends on the number of the molds to be fabricated.
4. Degas the mixture by applying a vacuum with the use of an in-house laboratory vacuum or vacuum pump in a vacuum desiccator for 1 hour.
5. Dab the surface of the mold with an adhesive tape to remove dust, and then pour the PDMS mixture to the mold. If bubbles are introduced in the process, repeat degassing in the vacuum desiccator. Bubbles trapped between pillars of the mold can be removed by poking them with a sharp needle.
   NOTE: The degassing process at room temperature should be done within 1 hour or less.
6. Cure the PDMS by heating at 80 °C for 2 hours. Allow the mold to cool down at room temperature.
7. Cut the boundary between the mold and PDMS with a blade and then peel off PDMS from the mold carefully.
8. Trim the PDMS part to fit the 22 mm x 22 mm coverslip and punch a hole at the inlet.
   NOTE: The protocol can be paused here.
9. Clean the PDMS part and 22 mm x 22 mm coverslip by wiping the surfaces with low-lint tissues and 70% ethanol. Then remove large dust particles by dabbing with an adhesive tape.
10. Sterilize the PDMS part and coverslip by either autoclaving (121 °C for 8 minutes wet, 15 minutes dry) or exposure to UV light for 1 hour.
11. Treat the bottom surface of the PDMS part and cover slip with corona discharge gun for 5 min in the tissue culture hood and then bond them together.
    CAUTION: Lay any non-conducting material such as polystyrene foam on the bottom and remove all conducting materials during this process. Inject collagen into the device immediately, within 5 minutes, after the treatment to increase the bonding between collagen gel and glass coverslip.

2. Cell preparation: primary mammary organoid isolation

NOTE: The details of mammary organoid isolation can be found in a previous work¹¹. Any kind of single cell or organoid can be prepared according to their own isolation/detachment protocols.

1. Mince the mammary gland tissue of the mice with a scalpel until the tissue relaxes and shake it for 30 min at 37 °C in 50 mL of collagenase/trypsin solution (10 mL per mouse) in DEME/F12 supplemented with 0.1 g of trypsin, 0.1 g of collagenase, 5 mL of FBS, 250 μL of 1 μg/mL insulin, and 50 μL of 50 μg/mL gentamicin.
2. Centrifuge the collagenase/trypsin solution at 1,250 x g for 10 min. Remove the supernatant by aspiration. Disperse cells in 10 mL of DMEM/F12, and centrifuge at 1,250 x g for 10 min. After removing DMEM/F12 by aspiration, re-suspend cells in 4 mL of DMEM/F12 supplemented with 40 μL of DNase (2 units/μL).
3. Shake the DNase solution by hand for 2-5 min and then centrifuge at 1,250 x g for 10 min.
4. Separate organoids from single cells through four differential centrifugations (pulse to 1,250 x g and stop the centrifuge 3-4 s after it reaches the intended speed). Between each pulse, aspirate the supernatant and resuspend the pellet in 10 mL of DMEM/F12.
5. Re-suspend the final pellet in the desired amount of growth medium of DMEM/F12 with 1% penicillin/streptomycin and 1% insulin- transferrin-selenium-X for collagen preparation.

3. Collagen preparation and injection

NOTE: Other types of ECM gels can be prepared according to their own gelation protocols.

1. Prepare collagen type I solution (3.78 mg/mL), 10x DMEM, 1 N NaOH solution, normal growth medium on ice.
   NOTE: Keep on ice until the procedure is completed.
2. Add 6 μL of 1 N NaOH solution, 200 μL of growth medium and 50 μL of 10x DMEM into a pre-chilled microcentrifuge tube and mix the solution thoroughly.
3. Add 425 μL of collagen into the pre-mixed solution and mix thoroughly by pipetting.
4. Centrifuge the collagen mixture briefly (less than 5 seconds) to separate and remove bubbles from the mixture.
5. Keep the neutralized collagen solution on ice for 1 hour to induce fiber formation.
6. Add 50 μL of cell suspension into the collagen mixture and mix thoroughly.
   NOTE: The final collagen concentration is 2 mg/mL.
7. Inject the solution using 200 μL pipet through the inlet of the PDMS device until the whole chamber is filled in. If the collagen mixture overflows through the gaps of pillars, cut out the excess collagen gel with sharp surgical blade after gelation.
8. Keep the device in the incubator at 37 °C with 5% CO₂ for 1 hour to induce gelation.
9. Fill the reservoirs on both sides with growth medium and keep the device in the incubator at 37 °C with 5% CO2 until confocal imaging is performed.

4. 3D imaging and quantification

1. Install a live-cell imaging culture chamber to a confocal microscope and pre-set the temperature and CO₂ to 37 °C and 5%, respectively. To get humidity up, add water in the reservoir of the chamber and place wet wipes in the chamber if necessary.
2. Place the PDMS device in the chamber and add EGF to one of the reservoir as a ‘source’ of the EGF in the gradient formation. The other reservoir will serve a ‘sink’ needed for development of the spatially graded EGF distribution. 2.5 nM of EGF was added in the sink for making the gradient of 0.5 nM/mm in this study.
3. Set the range of Z-stack covering the volume of organoids of interest and start the imaging.
   CAUTION: To avoid phototoxicity, try a lower laser intensity in confocal imaging or use multi-photon imaging techniques.
4. Reconstruct a 3D image from 2D image stacks using either commercial software or a custom-made program. Measure the length and angle of branches extending from the organoids or migration of individual cells. Here, perform quantification by drawing a freehand outline around the organoid body using ImageJ.

Results

EGF is an essential regulator of branching morphogenesis in mammary glands and a critical chemoattractant guiding the migration of breast epithelial cells in invasive cancer growth. We used the mesoscopic fluidic devices described above to study the response of cells to defined EGF gradients (Figure 1A,B)¹⁰. The device yields a culture area 5 mm wide, 10 mm long, and 1 mm tall. The sides of the culture area are separat...

Discussion

The fabrication of PDMS molds was performed using a commercial 3D printing service, but can also be accomplished by a high end 3D printer in-house. Among various 3D fabrication methods, stereolithography is recommended for high resolution mold generation. Because PDMS curing occurs at a high temperature (80 °C), the materials should be sufficiently thermally resistant, which should be explicitly specified, if printing is outsourced. A thermal post-cure can be discussed with the printing service company to increase t...

Materials

Name	Company	Catalog Number	Comments
22mm x 22mm coverslip	Fisher Scientific	12-542-B
Collagen I, Rat	Fisher Scientific	CB-40236
Collagenease	Sigma-Aldrich	C5138
COMSOL Multiphysics 4.2	COMSOL Inc		Used for simulating diffusion dynamics
10x DMEM	Sigma-Aldrich	D2429
DEME/F12	Thermo Fisher	11330032
DNase	Sigma-Aldrich	D4623
EGF Recombinant Mouse Protein	Thermo Fisher	PMG8041
Fetal Bovine Serum (FBS)	Life technologies	16140-071
Fiji-ImageJ			Used for measuring branching length and angles
Gentamicin	GIBCO	5750-060
IMARIS	Bitplane
Insulin	Sigma-Aldrich	19278
Insulin-Transferrin-Selenium-X	GIBCO	51500
Low-lint tissue	Kimberly-Clark Professional	Kimtech wipe
Mold Material	Proto labs	Accura SL5530
Mold printing equipment	Proto labs	Stereolithogrphy	Maximum dimension: 127mm x 127mm x 63.5mm, Layer thnickness: 0.0254mm
Mold printing Service	Proto labs	Custom	https://www.protolabs.com/
NaOH	Sigma-Aldrich	S2770
Penicillin/Streptomycin	VWR	16777-164P
Spinning-disk confocal microscope	Solamere Technology Group
Sylgard 184	Electron Microscopy Sciences	184 SIL ELAST KIT	PDMS kit
Trypsin	Sigma-Aldrich	T9935