An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

Summary

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

Abstract

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells.

Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

Introduction

In vivo, cells communicate with each other either directly (cell contact) or indirectly (by secretion of molecules). To study cell communication, different co-culture models can be developed, such as direct co-culture (the different cell types are in direct interaction in the same well) and compartmentalized co-culture (the different cell types are in indirect interaction in different compartments of a culture system)¹. Moreover, conditioned media can be used for co-culture systems, where the indirect interaction is enabled by secreted molecules contained in the conditioned media of an effector cell type being transferred to a responder cell type¹.

In the case of paracrine cell communication studies, indirect co-culture systems provide models that strongly reflect cell interactions in vivo. Indirect co-culture systems have been developed and commercialized, allowing the establishment of indirect co-culture models^2,3. Unfortunately, most indirect co-culture systems provide only two compartments. Other indirect co-culture systems provide multiple compartments, but they are less scalable as compared to the system reported in the present manuscript. Some of them do not allow a classical visualization under a microscope, and they often present specific application methods. In several studies, the paracrine communication between different cell types is probed by the conditioned media model^4,5,6,7. This is an easier way of investigation compared to indirect co-culture systems because it does not necessitate specific methods or materials to be established¹. On the other hand, the preparation of conditioned media is time-consuming and provides information only on one-way cell signaling (effector to responder)¹.

In this paper, a new, simple way of investigating cell communication is proposed. Allowing the combination of several cell types in direct or indirect interaction and in 2D or 3D formats, the printed inserts present numerous advantages for setting up co-culture models easily. Adapted to be placed into the wells of 6-well plates, the 3D-printed insert is circular and allows separation of the well into four compartments (two large compartments and two small compartments; Figure 1A). The 3D-printed inserts are characterized by the absence of a bottom. Thus, the cells are in direct contact with the plate on which the insert is placed. In addition, each compartment can be coated independently of the others. Moreover, the cell behavior can be easily followed under optical microscopes. The presence of communication windows in each wall of the insert allows the addition, at the optimal time, of a common medium to perform different experiments of co-culture. Numerous combinations of co-culture can be performed to study direct and/or indirect communication between several cell types. For example, a model of indirect co-culture between four different cell types in monolayer and/or in 3D (spheroids) can be designed. A combination of direct and indirect co-culture models can also be performed by mixing different cell types in the same compartment. The effect of complex structures (organoids, tissue explant, etc.) on different cell types could be another example of models that can be made. Moreover, the 3D-printed inserts are compatible with cell biology functional assays (proliferation, migration, pseudotube formation, differentiation, etc.) and with biochemistry tests (the extraction of DNA, RNA, protein, lipids, etc.). Finally, the 3D-printed inserts provide a wide range of experimental schemes of co-culture models with the possibility to combine simultaneously different assays in the same experiment in the different compartments.

Some capacities of the 3D-printed inserts are presented to validate them as a rapid and easy-to-use co-culture model. In comparison to a previously published study conducted on paracrine cell communication, the ability of the 3D-printed inserts to be a valuable co-culture model is demonstrated. To assess this point, the regulation of endothelial cell proliferation and migration by keratinocytes was compared between the 3D-printed insert system and the classical system using conditioned media. The 3D-printed inserts allow for obtaining similar results rapidly as compared to the conventional system using conditioned media. Indeed, the 3D-printed inserts provide a robust model to study cell interactions in both directions without the necessity to produce conditioned media and with the possibility to perform in parallel the proliferation and migration assays in the same experiment.

To conclude, in this paper, a new and ready-to-use model to study cell communication is proposed. Compatible with all adherent cell types, the 3D-printed inserts allow for performing numerous combinations of co-culture that aim at being closer to in vivo conditions.

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Protocol

NOTE: The 3D inserts (Figure 1A) are commercially procured and are printed using a photopolymer resin that is biocompatible with cell culture and autoclaving (see the Table of Materials). In this section, a detailed protocol to establish the co-culture model by inserts is described (see Figure 1B). Some examples of applications are also provided.

1. Placement of the sterilized insert in the 6-well plates

1. Mix the two components provided in the kit (see Table of Materials), food silicon (reagent A) and catalyst (reagent B), in a ratio of 10:1 (v/v) according to the manufacturer's instructions. (Figure 1Ba). Use a 70% ethanol sterilized spatula to mix the two components.
   NOTE: The volume of the silicon mixture to be prepared depends on the number of inserts to be fixed. An excess of catalyst can result in the silicon mixture solidifying too fast.
2. Apply the inserts on the silicon mix with tweezers so that the mixture is homogeneously distributed at the bottom edge of the inserts (Figure 1Bb). Place the inserts into the wells of a 6-well plate and apply gentle pressure on the inserts to ensure that the inserts are in close contact with the plate to avoid any leakage of the cell culture medium (Figure 1Bc).
3. Put the plate at 37 ˚C to support the solidification process of the silicon for 1 h.
   NOTE: The time of solidification depends on the quantity of catalyst that has been added.
4. After solidification, sterilize the plates with the inserts by immersing them in a 70% ethanol bath for 30 min to 1 h.
5. Discard the alcohol by pipetting and let the plate dry (lid open) overnight in a cell culture hood.
   ​NOTE: The alcohol must be fully evaporated before performing the next step to avoid cell fixation and toxicity. The ready-to-use plate containing the inserts can be stored for several days before use in dry and sterile conditions at room temperature.

2. Coatings such as poly-L-lysine, or poly-HEMA (Optional step)

NOTE: Coating is not performed in this experiment, and the steps are provided to inform regarding the possibility of coating the inserts.

1. Prepare the coating solution according to the manufacturer's instructions.
2. Consider a volume of 200-400 µL for the largest compartments and a volume of 50-100 µL for the smallest compartments for the coating of the insert. Add the coating solution to the individual compartments.
3. Let the coating dry as indicated by the manufacturer under sterile conditions.

3. Seeding of the cells

1. Culture the keratinocytes of the outer root sheath (KORS) and human dermal microvascular endothelial cells (HDMECs) as recommended by the manufacturer. Prepare the cell suspension once the cells reach confluency.
   1. Discard the medium from the flasks and add 5 mL of trypsin to detach the cells (see Table of Materials).
   2. Place the flask containing the KORS at 37 °C with 5% CO₂ for 5 min. Incubate the flask containing the HDMECs for 5 min at room temperature.
   3. Mix the trypsin solution containing the KORS with 5 mL of mesenchymal stem cell medium (MSC_M) supplemented with 5% fetal bovine serum (FBS) and growth factors (basal medium with supplement kit, see Table of Materials). Mix the trypsin solution containing the HDMECs with 5 mL of endothelial cell medium (EC_M) supplemented with 5% FBS and growth factors (see Table of Materials).
   4. Transfer the cells into a 15 mL of sterile conical tube. Centrifuge the KORS and HDMEC suspensions at 200 x g for 5 min at room temperature.
   5. Discard the supernatant and resuspend the cells in 2 mL of the respective medium.
   6. Mix 10 µL of the cell suspension with 10 µL of trypan blue dye (see Table of Materials).
   7. Add 10 µL of the mix into the chamber of the counting slide.
   8. Insert the counting slide into the cell counting system (see Table of Materials) and detect the cell number and the viability.
   9. Prepare the cell suspension at a concentration of 0.5 x 10⁵ cells/mL in the respective medium.
      NOTE: If the different cell types require different times of adherence and/or to reach the ideal confluency, the cell seeding can be made at different time points according to the cell types.
2. Distribute 800 µL to 1 mL of the cell suspension into the individual large compartments and 100-150 µL into the individual small compartments with a pipette.
   1. Seed the KORS at 1 x 10⁵ cells/mL in MSC_M supplemented with 5% FBS and growth factors in one of the big compartments.
   2. Seed HDMEC in EC_M supplemented with 5% FBS and growth factors at 2.5 x 10³ cells/mL in the small compartments and at 1.25 x 10⁵ cells/mL in the other big compartment.
      NOTE: The proliferation assay can be performed in the small compartments, and the migration and viability assays can be performed in the big compartments. The concentration of the cells seeded has been optimized according to the manufacturer's recommendations. This concentration allows for good viability of the cells after 24-48 h of incubation.
3. Place the cell culture plate at 37 ˚C with 5% CO₂ or under the usual conditions to allow for cell adhesion and/or maturation.
   NOTE: Incubate the cells for 24-48 h without changing the media. If needed, the medium of the compartments can be changed independently.

4. Implementation of the co-culture ( Figure 1Bd)

NOTE: The implementation of the co-culture corresponds to the time when the common medium is added. The addition of a unique medium for all the cell types in the different compartments provides the possibility of paracrine communication between the cells due to the presence of communication windows (ovoid holes in the walls of the 3D-printed inserts). To implement the co-culture, follow steps 4.1-4.3.

1. Empty the culture media of the different compartments of the inserts using a pipette. In case of a 3D spheroid cell culture, discard the medium very gently.
2. Rinse the cells with 1 mL of 37 °C warm PBS in the big compartments and 200 µL of 37 °C warm PBS in the small compartments. Ensure gentle washing to not detach the cells.
3. Add 3 mL of a common medium selected to be compatible for all cell types.
   NOTE: In this study, the basal EC_M (without FBS and growth factors) is used. Make sure that the medium covers all the compartments (level above the communication windows [ovoid holes in the walls], as shown in Figure 1A).
4. Place the plate containing the co-cultures at 37 ˚C with 5% CO₂ for 24-48 h.

5. Cell observation, counting, and scraping

1. Observe the morphology and count the cell number of the different compartments during the experiment under an optical microscope after 24-48 h of incubation.
   NOTE: The expected number of cells depends on the cell types used (size, morphology, etc.) and on the compartment size. In this experiment, between 0.5 x 10⁶-1 x 10⁶ KORS/mL and between 0.25 x 10⁶-0.75 x 10⁶ HDMECs/mL are counted in the big compartments.
2. Detach the cells using trypsin for cell suspension counting.
   NOTE: For the cell detachment solution, consider a volume of 100-500 µL for the largest compartments and a volume of 10-50 µL for the smallest compartments.
3. Check the viability by using trypan blue staining (see steps 3.1.6-3.1.8).

6. Insert cleaning for recycling

1. Remove the inserts from the 6-well plate at the end of the experiment by a slight twist (Figure 1Be).
2. Remove the silicon from the inserts by pulling it off. If necessary, use a spatula to remove the remaining silicon stuck to the walls of the inserts (Figure 1Bf).
3. Wash the inserts with conventional cell culture detergents and cleaning materials (example provided in the Table of Materials).
4. Sterilize the inserts for future use in an autoclave (solid cycle, 121 ˚C for 20 min) or by immersing them in a 70% ethanol bath for 1 h.
   ​NOTE: From this step, two examples of possible assays (proliferation and migration assays) are described using the 3D-printed inserts (Figure 1Bd).

7. Cell proliferation assay - WST-1 assay

1. Follow steps 1-3.1 to culture the cells.
   NOTE: Follow the step in this section to implement the co-culture system in order to investigate the effect of the KORS secretome on HDMEC proliferation. KORS cells are used to compare the data published previously⁸.
   1. Seed the KORS at 1 x 10⁵ cells/mL in mesenchymal stem cell medium (MSC_M) supplemented with 5% FBS and growth factors in the big compartments.
   2. Place the cell culture plate at 37 ˚C with 5% CO₂ for 24 h.
   3. Seed the HDMECs in endothelial cell medium (EC_M) supplemented with 5% FBS and growth factors at 2.5 x 10³ cells/mL in the small compartments.
   4. Place the cell culture plate at 37 °C with 5% CO₂ for 24-48 h.
   5. Discard the media from all the compartments and implement the co-culture system by the addition of 3 mL of non-supplemented basal EC_M (common cell culture medium for all the compartments of the 3D-printed inserts).
2. Dilute WST-1 in the EC_M common cell culture medium (1:10 final dilution) to prepare the WST-1 solution.
   ​NOTE: For the volume of WST-1 solution, prepare at least 500 µL of extra solution for the blank.
3. Discard the cell culture medium from the insert by pipetting.
4. Add the WST-1 solution to the selected compartments (150 µL for the small compartments and 600 µL for the big compartments). Put the plate at 37 ˚C with 5% CO₂.
5. After the optimal 30 min cell incubation time, transfer 100 µL of the incubation medium from each condition on a 96-well plate.
6. Assess the colorimetric reaction using a microplate reader between 420 nm and 480 nm.
   1. Put the plate on the microplate reader and click on the button edit a new protocol.
   2. Select the specific type of 96-well plate (see Table of Materials) used and the wavelength of 450 nm.
   3. Click on the button start measurement.

8. Cell migration assay - two migration chambers device

1. Follow steps 1-3.1 to culture the cells.
   NOTE: Follow the steps in this section to implement the co-culture system in order to investigate the effect of the KORS secretome on HDMEC proliferation.
   1. Seed the KORS at 1 x 10⁵ cells/mL in MSC_M supplemented with 5% FBS and growth factors in one of the big compartments.
   2. Place the cell culture plate at 37 ˚C with 5% CO₂ for 24 h.
   3. Put the two migration chambers device into the other large compartment of inserts.
   4. Seed the HDMECs in EC_M supplemented with 5% FBS and growth factors at 7 x 10⁵ cells/well of the two migration chambers device.
   5. Place the cell culture plate at 37 °C with 5% CO₂ for 24 h.
   6. Discard the media and the two migration chambers device after 24 h or at an optimal time according to the cell type that is migrating. Implement the co-culture system by the addition of 3 mL of non-supplemented basal EC_M.
      NOTE: Proceed carefully to not detach the cells. If the cells are really sensitive to detachment induced by the addition of common medium, pull off the two migration chambers device after the addition of the common medium.
2. Observe and take a picture of the surface covered by the cells at different time points under a phase-contrast microscope at 10x magnification.
   NOTE: The pictures are taken with a phase-contrast microscope (see Table of Materials). The pictures are saved in a USB key and then analyzed by the wound healing tool plugin of the software (see Table of Materials).
3. Measure the uncovered surface using classical quantitative analysis software.
   1. Open the image on the software and activate the wound healing tool available in the macro list.
   2. Click on the m button to measure the uncovered surface of the migration image.
   3. The uncovered surface is calculated according to the following formula:
      Percentage of coverage = ([mean area without cells at T0 − area without cells at time T]/mean area without cells at T0) × 100.
      NOTE: The 3D-printed inserts are totally adapted to most of the techniques of cell biology (such as the pseudotube formation assay, 3D cultures) and for biochemistry experiments (such as the extraction of DNA, RNA, protein).

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Results

In the present work, an optimized co-culture system was described to obtain robust, reliable, and significant data comparable to classical methods through the use of conditioned media. The 3D-printed inserts mimic the in vivo microenvironment conditions of different cell types in interaction by overcoming the difficulties and the time-consuming production of conditioned media upstream in the experiments. A previously published study has been reconducted to analyze the indirect interactions between two cell types...

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Discussion

Indirect cell communication is commonly investigated using conditioned media or co-culture system devices. Conditioned media preparation is time-consuming upstream in the experiments, and this method is restricted to one-sided effect analyses. The previous study by Colin-Pierre et al.⁸ using conditioned media was conducted on the indirect cell communication between two cell types (HDMECs and KORS). The data of this previous study demonstrated the effect of KORS conditioned media on HDMEC ...

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Materials

Name	Company	Catalog Number	Comments
Autoclave	Getinge	APHP	Solid cycle, 121 °C for 20 min
Biomed Clear	Formlabs	RS-F2-BMCL-01	Impression performed by 3D-Morphoz company (Reims, France)
Cell culture detergents	Tounett	A18590/0116
Cell Proliferation Reagent WST-1	Roche	11,64,48,07,001
Counting slide	NanoEnTek	EVE-050
Culture-Insert 2 Well in μ-Dish 35 mm	Ibidi	80206	two-migration chambers device.
Endothelial cell medium	ScienCell	1001	Basal medium +/- 25 mL of fetal bovine serum (FBS, 0025), 5 mL of endothelial cell growth supplement (ECGS, 1052), and 5 mL of penicillin/streptomycin solution (P/S, 0503).
EVE Automated cell counter	NanoEnTek	NESCT-EVE-001E
EVOS XL Core	Fisher Scientific	AMEX1200	10x of magnification
Food silicon reagent and catalyst kit	Artificina	RTV 3428 A and B	(10:1)
FORM 3B printer	Formlabs	PKG-F3B-WSVC-DSP-BASIC	Impression performed by 3D-Morphoz company
Human Dermal Microvascular Endothelial Cells (HDMEC)	ScienCell	2000
Keratinocytes of Outer Root Sheath (KORS )	ScienCell	2420
Macro Wound Healing Tool Software	ImageJ		Software used for the measurement of the uncovered surface (for migration assays)
Mesenchymal stem cell medium	ScienCell	7501	Basal medium +/-25 mL of fetal bovine serum (FBS, 0025), 5 mL of mesenchymal stem cell growth supplement (MSCGS, 7552), and 5 mL of penicillin/streptomycin solution (P/S, 0503)
Microplate reader SPECTRO star NANO	BMG Labtech		BMG LABTECH software
PBS	Promocell	C-40232	Without Ca2+ / Mg2+
Trypan Blue Stain	NanoEnTek	EBT-001
Trypsin / EDTA	Promocell	C-41020	Incubation of KORS at 37 °C with 5% CO2 for 5 min. Incubation of HDMECs for 5 min at room temperature
96-well plate Nunclon Delta Surface	Thermoscientific	167008