Name: an innovative 3d-printed insert designed to enable straightforward 2d and 3d cell cultures
Uploaded: 2023-01-06T13:35:00
Description: Watch this Scientific Journal Video about An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures at JoVE.com

This study was made in collaboration with BASF Beauty Care Solutions. Ms. Charlie Colin-Pierre is a BASF /CNRS-funded PhD fellow.
We wish to thank Mr. Mehdi Sellami for the conception of the 3D-printed inserts.

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</strong...

<ol>
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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 ...

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 models2,3. 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 model4,5,6,7. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Autoclave</td>
			<td>Getinge</td>
			<td>APHP</td>
			<td>Solid cycle, 121 &#176;C for 20 min</td>
		</tr>
		<tr>
			<td>Biomed Clear</td>
			<td>Formlabs</td>
			<td>RS-F2-BMCL-01</td>
			<td>Impression performed by 3D-Morphoz company (Reims, France)</td>
		</tr>
		<tr>
			<td>Cell culture detergents&#160;</td>
			<td>Tounett</td>
			<td>A18590/0116</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Proliferation Reagent WST-1</td>
			<td>Roche</td>
			<td>11,64,48,07,001</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slide</td>
			<td>NanoEnTek</td>
			<td>EVE-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture-Insert 2 Well in &#956;-Dish 35 mm</td>
			<td>Ibidi</td>
			<td>80206</td>
			<td>two-migration chambers device.&#160;</td>
		</tr>
		<tr>
			<td>Endothelial cell medium</td>
			<td>ScienCell</td>
			<td>1001</td>
			<td>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).</td>
		</tr>
		<tr>
			<td>EVE Automated cell counter&#160;</td>
			<td>NanoEnTek</td>
			<td>NESCT-EVE-001E&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL Core</td>
			<td>Fisher Scientific</td>
			<td>AMEX1200</td>
			<td>10x of magnification</td>
		</tr>
		<tr>
			<td>Food silicon reagent and catalyst kit</td>
			<td>Artificina</td>
			<td>RTV 3428 A and B&#160;</td>
			<td>(10:1)</td>
		</tr>
		<tr>
			<td>FORM 3B printer</td>
			<td>Formlabs</td>
			<td>PKG-F3B-WSVC-DSP-BASIC</td>
			<td>Impression performed by 3D-Morphoz company</td>
		</tr>
		<tr>
			<td>Human Dermal Microvascular Endothelial Cells (HDMEC)</td>
			<td>ScienCell</td>
			<td>2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocytes of Outer Root Sheath (KORS )</td>
			<td>ScienCell</td>
			<td>2420</td>
			<td></td>
		</tr>
		<tr>
			<td>Macro Wound Healing Tool Software</td>
			<td>ImageJ</td>
			<td></td>
			<td>Software used for the measurement of the uncovered surface (for migration assays)</td>
		</tr>
		<tr>
			<td>Mesenchymal stem cell medium&#160;</td>
			<td>ScienCell</td>
			<td>7501</td>
			<td>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)</td>
		</tr>
		<tr>
			<td>Microplate reader SPECTRO star NANO</td>
			<td>BMG Labtech</td>
			<td></td>
			<td>BMG LABTECH software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Promocell</td>
			<td>C-40232</td>
			<td>Without Ca2+ / Mg2+</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>NanoEnTek</td>
			<td>EBT-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin / EDTA</td>
			<td>Promocell</td>
			<td>C-41020</td>
			<td>Incubation of KORS at 37 &#176;C with 5% CO2 for 5 min. Incubation of HDMECs for 5 min at room temperature</td>
		</tr>
		<tr>
			<td>96-well plate Nunclon Delta Surface</td>
			<td>Thermoscientific</td>
			<td>167008</td>
			<td></td>
		</tr>
	</tbody>
</table>

an innovative 3d-printed insert designed to enable straightforward 2d and 3d cell cultures

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.

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...

Watch this Scientific Journal Video about An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures at JoVE.com

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

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.

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...

This protocol can be used to investigate numerous culture studies and provide a new tool to investigating direct cell communication. This new device saves significant time in establishing a culture model and is applicable to different cell types requiring a specific coating or not. Indeed, the four compartment of the 3D-printed inserts, allow to culture different cell types in monolayer, or in 3D in the same way with different combinations.The 3D-printed inserts are more durable, flexible, scalable, and can be used to design experimental models for the studies of physiopathologies, such as immunology, or androgenesis. To begin, mix the two components, food silicon, and catalyst provided in the kit in a ratio of 10:1, using a 70%ethanol sterilized spatula according to the manufacturer's instructions. Apply the inserts on the silicone mix with tweezers so that the mixture is homogenously distributed at the bottom edge of the insert.Place the inserts into the wells of a six well plate and apply gentle pressure to ensure that the inserts are in close contact with the plate to avoid any leakage of the cell culture medium. Put the plate at 37 degrees Celsius to support the solidification process of the silicon for one hour. After solidification, sterilize the plates with the inserts by immersing them in a 70%ethanol bath for 30 minutes to one hour.Next, discard the alcohol by pipetting, and let the plate dry overnight in a cell culture hood. To culture the keratinocytes of the outer root sheath and human dermal microvascular endothelial cells, discard the medium from the flasks and add five milliliters of trypsin to detach the cells. Place the flask containing the keratinocytes of the outer root sheath at 37 degrees Celsius with 5%carbon dioxide for five minutes, and incubate the flask containing the human dermal microvascular endothelial cells for five minutes at room temperature.In a 15 milliliter sterile conical tube, mix the trypsin solution containing the keratinocytes of the outer root sheath with five milliliters of mesenchymal stem cell medium supplemented with 5%FBS and growth factors and the human dermal microvascular endothelial cells with five milliliters of endothelial cell medium supplemented with 5%FBS and growth factors. Centrifuge the keratinocytes of the outer root sheath and human dermal microvascular endothelial cell suspensions at 200 G for five minutes at room temperature. After discarding the supernatant, resuspend the cells in two milliliters of their respective mediums.Mix 10 microliters of the cell suspension with 10 microliters of trypan blue dye, and add 10 microliters of the mix into the chamber of the counting slide. Insert the counting slide into the cell counting system and detect the cell number and viability. After preparing the cell suspension at a concentration of 50, 000 cells per milliliter in the respective medium, distribute 800 microliters to one milliliter of the cell suspension into the individual large compartments, and 100 to 150 microliters into the individual small compartments with a pipette.See the keratinocytes in mesenchymal stem cell medium supplemented with 5%FBS and growth factors and human dermal microvascular endothelial cells in endothelial cell medium, supplemented with 5%FBS and growth factors in the small and other big compartments. Place the cell culture plate at 37 degrees Celsius with 5%carbon dioxide, or under the usual conditions, to allow for cell adhesion and or maturation. Empty the culture media of the different compartments of the inserts using a pipette.Then rinse the cells with one milliliter of 37 degrees Celsius warm PBS in the big compartments, and 200 microliters in the small compartments. Add three milliliters of a common medium selected to be compatible for all cell types. Place the plate containing the co-cultures at 37 degrees Celsius with 5%carbon dioxide for 24 to 48 hours.Detach the cells using trypsin for cell suspension counting, and check the viability using trypan blue staining. After observing the morphology, count the cell number of the different compartments during the experiment under an optical microscope after 24 to 48 hours of incubation. First, remove the inserts from the six well plate at the end of the experiment by a slight twist, and then remove the silicon from the inserts by pulling it off.Wash the inserts with conventional cell culture detergents and cleaning materials. Then sterilize the inserts for future use in an autoclave, or by immersing them in a 70%ethanol bath for one hour. The phenotype and the cell viability were compared, where 90%viability was observed in the wells of the six well plates, and at 91%was observed in the 3D-printed inserts for keratinocytes of the outer root sheath.The viability of human dermal microvascular endothelial cells was 85%in the wells of the six well plate, whereas 86%in the 3D-printed inserts. The indirect cell communications between keratinocytes and endothelial cells in the 3D-printed inserts demonstrated that in the presence of keratinocytes in the inserts the human dermal microvascular endothelial cells'proliferation significantly increased by 1.5-fold after 24 hours, and by 3.1-fold after 48 hours compared to the control. However, the keratinocyte of the outer root sheath condition medium was shown to significantly increase human dermal microvascular endothelial cell proliferation by 1.5-fold after 24 hours, and by 2.1-fold after 48 hours.The 3D-printed insert co-culture model was tested to determine the effect of keratinocytes on human dermal microvascular endothelial cell migration. In the control condition, endothelial cells migrated and covered approximately 44%of the wound area after 24 hours. In the presence of keratinocytes, a significant increase in human dermal microvascular endothelial cell migration was observed, which shows that 43%67%and 99%of the wound area was covered after three, 12, and 24 hours respectively.Similarly, the migration of HDMECs increases in presence of keratinocytes of the outer root sheath condition medium. It is crucial to ensure the sealing of the insert to the plate, in order to prevent the leakage of the medium of cells from one compartment to another. Several applications could be made, such as proliferation, migration, sedative formation assays DNA, RNA, protein and other analysis.

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