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
<ol>
	<li>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&#39;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.</li>
	<li>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).</li>
	<li>Put the plate at 37 &#730;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.</li>
	<li>After solidification, sterilize the plates with the inserts by immersing them in a 70% ethanol bath for 30 min to 1 h.</li>
	<li>Discard the alcohol by pipetting and let the plate dry (lid open) overnight in a cell culture hood. 
	&#8203;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.</li>
</ol>
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.
<ol>
	<li>Prepare the coating solution according to the manufacturer&#39;s instructions.</li>
	<li>Consider a volume of 200-400 &#181;L for the largest compartments and a volume of 50-100 &#181;L for the smallest compartments for the coating of the insert. Add the coating solution to the individual compartments.</li>
	<li>Let the coating dry as indicated by the manufacturer under sterile conditions.</li>
</ol>
3. Seeding of the cells
<ol>
	<li>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.
	<ol>
		<li>Discard the medium from the flasks and add 5 mL of trypsin to detach the cells (see Table of Materials).</li>
		<li>Place the flask containing the KORS at 37 &#176;C with 5% CO2 for 5 min. Incubate the flask containing the HDMECs for 5 min at room temperature.</li>
		<li>Mix the trypsin solution containing the KORS with 5 mL of mesenchymal stem cell medium (MSCM) 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 (ECM) supplemented with 5% FBS and growth factors (see Table of Materials).</li>
		<li>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.</li>
		<li>Discard the supernatant and resuspend the cells in 2 mL of the respective medium.</li>
		<li>Mix 10 &#181;L of the cell suspension with 10 &#181;L of trypan blue dye (see Table of Materials).</li>
		<li>Add 10 &#181;L of the mix into the chamber of the counting slide.</li>
		<li>Insert the counting slide into the cell counting system (see Table of Materials) and detect the cell number and the viability.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Distribute 800 &#181;L to 1 mL of the cell suspension into the individual large compartments and 100-150 &#181;L into the individual small compartments with a pipette.
	<ol>
		<li>Seed the KORS at 1 x 105 cells/mL in MSCM supplemented with 5% FBS and growth factors in one of the big compartments.</li>
		<li>Seed HDMEC in ECM 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&#39;s recommendations. This concentration allows for good viability of the cells after 24-48 h of incubation.</li>
	</ol>
	</li>
	<li>Place the cell culture plate at 37 &#730;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.</li>
</ol>
4. Implementation of the co-culture (&#160;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.
<ol>
	<li>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.</li>
	<li>Rinse the cells with 1 mL of 37 &#176;C warm PBS in the big compartments and 200 &#181;L of 37 &#176;C warm PBS in the small compartments. Ensure gentle washing to not detach the cells.</li>
	<li>Add 3 mL of a common medium selected to be compatible for all cell types. 
	NOTE: In this study, the basal ECM (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).</li>
	<li>Place the plate containing the co-cultures at 37 &#730;C with 5% CO2 for 24-48 h.</li>
</ol>
5. Cell observation, counting, and scraping
<ol>
	<li>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.</li>
	<li>Detach the cells using trypsin for cell suspension counting. 
	NOTE: For the cell detachment solution, consider a volume of 100-500 &#181;L for the largest compartments and a volume of 10-50 &#181;L for the smallest compartments.</li>
	<li>Check the viability by using trypan blue staining (see steps 3.1.6-3.1.8).</li>
</ol>
6. Insert cleaning for recycling
<ol>
	<li>Remove the inserts from the 6-well plate at the end of the experiment by a slight twist (Figure 1Be).</li>
	<li>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).</li>
	<li>Wash the inserts with conventional cell culture detergents and cleaning materials (example provided in the Table of Materials).</li>
	<li>Sterilize the inserts for future use in an autoclave (solid cycle, 121 &#730;C for 20 min) or by immersing them in a 70% ethanol bath for 1 h. 
	&#8203;NOTE: From this step, two examples of possible assays (proliferation and migration assays) are described using the 3D-printed inserts (Figure 1Bd).</li>
</ol>
7. Cell proliferation assay - WST-1 assay
<ol>
	<li>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.
	<ol>
		<li>Seed the KORS at 1 x 105 cells/mL in mesenchymal stem cell medium (MSCM) supplemented with 5% FBS and growth factors in the big compartments.</li>
		<li>Place the cell culture plate at 37 &#730;C with 5% CO2 for 24 h.</li>
		<li>Seed the HDMECs in endothelial cell medium (ECM) supplemented with 5% FBS and growth factors at 2.5 x 103 cells/mL in the small compartments.</li>
		<li>Place the cell culture plate at 37 &#176;C with 5% CO2 for 24-48 h.</li>
		<li>Discard the media from all the compartments and implement the co-culture system by the addition of 3 mL of non-supplemented basal ECM (common cell culture medium for all the compartments of the 3D-printed inserts).</li>
	</ol>
	</li>
	<li>Dilute WST-1 in the ECM common cell culture medium (1:10 final dilution) to prepare the WST-1 solution. 
	&#8203;NOTE: For the volume of WST-1 solution, prepare at least 500 &#181;L of extra solution for the blank.</li>
	<li>Discard the cell culture medium from the insert by pipetting.</li>
	<li>Add the WST-1 solution to the selected compartments (150 &#181;L for the small compartments and 600 &#181;L for the big compartments). Put the plate at 37 &#730;C with 5% CO2.</li>
	<li>After the optimal 30 min cell incubation time, transfer 100 &#181;L of the incubation medium from each condition on a 96-well plate.</li>
	<li>Assess the colorimetric reaction using a microplate reader between 420 nm and 480 nm.
	<ol>
		<li>Put the plate on the microplate reader and click on the button edit a new protocol.</li>
		<li>Select the specific type of 96-well plate (see Table of Materials) used and the wavelength of 450 nm.</li>
		<li>Click on the button start measurement.</li>
	</ol>
	</li>
</ol>
8. Cell migration assay - two migration chambers device
<ol>
	<li>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.
	<ol>
		<li>Seed the KORS at 1 x 105 cells/mL in MSCM supplemented with 5% FBS and growth factors in one of the big compartments.</li>
		<li>Place the cell culture plate at 37 &#730;C with 5% CO2 for 24 h.</li>
		<li>Put the two migration chambers device into the other large compartment of inserts.</li>
		<li>Seed the HDMECs in ECM supplemented with 5% FBS and growth factors at 7 x 105 cells/well of the two migration chambers device.</li>
		<li>Place the cell culture plate at 37 &#176;C with 5% CO2 for 24 h.</li>
		<li>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 ECM. 
		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.</li>
	</ol>
	</li>
	<li>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).</li>
	<li>Measure the uncovered surface using classical quantitative analysis software.
	<ol>
		<li>Open the image on the software and activate the wound healing tool available in the macro list.</li>
		<li>Click on the m button to measure the uncovered surface of the migration image.</li>
		<li>The uncovered surface is calculated according to the following formula: 
		Percentage of coverage = ([mean area without cells at T0 &#8722; area without cells at time T]/mean area without cells at T0) &#215; 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).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Regier, M. C., Alarid, E. T., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+towards+understanding+heterotypic+interactions+in+multi-culture+models+of+breast+cancer.">Progress towards understanding heterotypic interactions in multi-culture models of breast cancer.</a> Integrative Biology. 8 (6), 684-692 (2016).</li><li>Do&#287;an, A., Demirci, S., Apdik, H., Apdik, E. A., &#350;ahin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+isolation+from+pulp+tissue+and+co-culture+with+cancer+cells+to+study+their+interactions.">Mesenchymal stem cell isolation from pulp tissue and co-culture with cancer cells to study their interactions.</a> Journal of Visualized Experiments. (143), e58825 (2019).</li><li>Lin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA+UBE2CP3+enhances+HCC+cell+secretion+of+VEGFA+and+promotes+angiogenesis+by+activating+ERK1/2/HIF-1&#945;/VEGFA+signalling+in+hepatocellular+carcinoma.">Long non-coding RNA UBE2CP3 enhances HCC cell secretion of VEGFA and promotes angiogenesis by activating ERK1/2/HIF-1&#945;/VEGFA signalling in hepatocellular carcinoma.</a> Journal of Experimental &amp; Clinical Cancer Research. 37 (1), 113 (2018).</li><li>Soares, N. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropoelastin+promotes+the+formation+of+dense,+interconnected+endothelial+networks.">Tropoelastin promotes the formation of dense, interconnected endothelial networks.</a> Biomolecules. 11 (9), 1318 (2021).</li><li>Mercatali, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+human+preclinical+model+of+osteoclastogenesis+from+peripheral+blood+monocytes+co-cultured+with+breast+cancer+cell+lines.">Development of a human preclinical model of osteoclastogenesis from peripheral blood monocytes co-cultured with breast cancer cell lines.</a> Journal of Visualized Experiments. (127), e56311 (2017).</li><li>Orona, N. S., Astort, F., Maglione, G. A., Yakisich, J. S., Tasat, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+indirect+effect+of+air+particles+exposure+induce+Nrf2-dependent+cardiomyocyte+cellular+response+in+vitro.">Direct and indirect effect of air particles exposure induce Nrf2-dependent cardiomyocyte cellular response in vitro.</a> Cardiovascular Toxicology. 19 (6), 575-587 (2019).</li><li>Colin-Pierre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glypican-1/HGF/C-met+and+glypican-1/VEGF/VEGFR2+ternary+complexes+regulate+hair+follicle+angiogenesis.">The glypican-1/HGF/C-met and glypican-1/VEGF/VEGFR2 ternary complexes regulate hair follicle angiogenesis.</a> Frontiers in Cell and Developmental Biology. 9, 781172 (2021).</li><li>Toubal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NC1+domain+of+type+XIX+collagen+inhibits+melanoma+cell+migration.">The NC1 domain of type XIX collagen inhibits melanoma cell migration.</a> European Journal of Dermatology. 20 (6), 712-718 (2010).</li><li>Wu, A. -. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmin+releases+the+anti-tumor+peptide+from+the+NC1+domain+of+collagen+XIX.">Plasmin releases the anti-tumor peptide from the NC1 domain of collagen XIX.</a> Oncotarget. 6 (6), 3656-3668 (2015).</li><li>Martinotti, S., Ranzato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scratch+wound+healing+assay.">Scratch wound healing assay.</a> Methods in Molecular Biology. 2109, 225-229 (2020).</li><li>Cattin, S., Ramont, L., R&#252;egg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+in+vivo+validation+of+a+three-dimensional+multi-cellular+culture+model+to+study+heterotypic+interactions+in+colorectal+cancer+cell+growth,+invasion+and+metastasis.">Characterization and in vivo validation of a three-dimensional multi-cellular culture model to study heterotypic interactions in colorectal cancer cell growth, invasion and metastasis.</a> Frontiers in Bioengineering and Biotechnology. 6, 97 (2018).</li><li>Ma, Y. 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The authors declare no competing financial interests.

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 ...

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Research

JoVE Journal

Biology

1.3K Views.  Université de Reims Champagne-Ardenne. 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.

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

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis. This has traditionally been done by netting foragers at nest entrances or on flowers, chilling the bees on ice, and then removing the pollen loads from the corbiculae with forceps or a brush. This method is time and labor intensive, may alter normal foraging behavior, and can result in stinging incidents for the worker performing the task. Pollen traps, such as those used on honey bee hives, collect pollen by dislodging corbicular pollen loads from the legs of workers as they pass through screens at the nest entrance. Traps can remove a large quantity of pollen from returning forager bees with minimal labor, yet to date no such trap is available for use with bumble bee colonies. Workers within a bumble bee colony can vary in size making size selection of entrances difficult to adapt this mechanism to commercially reared bumble bee hives. Using 3D printing design programs, we created a pollen trap that successfully removes the corbicular pollen loads from the legs of returning bumble bee foragers. This method significantly reduces the amount of time required by researchers to collect pollen from bumble bee foragers returning to the colony. We present the design, results of pollen removal efficiency tests, and suggest areas of modifications for investigators to adapt traps to a variety of bumble bee species or nest box designs.

A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

We present a non-lethal and automated mechanism to collect pollen from bumble bee (Bombus) workers returning to a hive. Instructions for producing, preparing, installing and using the devices are included. By using 3D-printed objects, modification to the design was timely, efficient and allowed for quick turnaround for testing.

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis.  ...

Using a 3D Printed Holder for Bulk Electroacupuncture in Small Animals

Bulk Electroacupuncture Operation for Mice or Young Rats

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering its promotion. The mechanistic study requires mouse or rat models to address this issue. However, these animals are not obedient to the experimental process, which is time-consuming. To solve these problems, we designed a 3D-printed small animal body bulk fixator to improve the efficiency of EA's animal experiments. This video shows in detail how to use the fixator to perform bulk EA on mice or young rats. For the selection of acupoints, the anterior oblique line of vertex temporal (MS6 head) and Tianshu point (ST25 belly) were chosen to verify the effect of the fixation device with prone positioning and supine positioning. Using the 3D-printed small animal holder allows multiple rodents to be immobilized and treated simultaneously, reducing the time and resources required for the experiment. This technique could be applied to other animal models by 3D printing different sizes and could potentially be used for various fixing conditions. The device is beneficial for the promotion of experimental scientific research in EA.

Author Spotlight: Using a 3D-Printed Holder for Simultaneous Multiple Electroacupuncture Operation

Here, we present a protocol for efficient and rapid electroacupuncture (EA) in mice or young rats using a three-dimensional (3D) printed holder. This technique allows simultaneous operation on multiple animals, saving time and increasing experimental efficiency.

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering ...

Purification of Human iPSC-Derived Hepatocytes by Mitochondrial-ALCAM Sorting

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% &plusmn; 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.

Author Spotlight: Advancing Hepatocyte Purification from Human Induced Pluripotent Stem Cells for Regenerative Medicine

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166) staining.

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...

Innovating an Improved Cell Scraper for Cell Wounding by User Requirements Analysis

A Tool to Automatically Create Stable and Reproducible Cell-free Gaps for Improving the Reliability of Cell Wound Healing Assay

The reliability of cellular migration measurement in wound healing assays is frequently undermined by the prevalent methodological instability, i.e., tip-based method. We introduce an innovative instrument designed to address these limitations. Our novel cell scraper surpasses the current approach, generating a more consistent and stable cell-free gap. Repeated biological experiments reveal that the cell-free gap produced by the cell scraper exhibits straighter edges and uniform size and shape compared to the tip-based technique (p &lt; 0.05). In terms of product design, the cell scraper boasts a refined color scheme suited for laboratory environments, enhancing the monitoring of experimental outcomes, and permits sterilization through autoclaving for reuse. Notably, after treatment, the cell scraper demonstrates a negligible effect on cell viability and proliferation (97.31% and 24.41%, respectively). Conversely, the tip-based method yields lower cell viability (91.37%) and proliferation (18.79%). This investigation presents the cell scraper as a novel, reusable device capable of generating reproducible cell-free gaps while preserving cellular viability, thereby augmenting the reliability of wound healing assays in comparison to existing techniques.

Through the incorporation of interaction experience design and user requirements analysis, we introduce an innovative cell scraper that enhances cellular wound healing assays in terms of reproducibility, dependability, practicality, cellular integrity, and user experience.

The reliability of cellular migration measurement in wound healing assays is frequently undermined by the prevalent methodological instability, i.e., ...

3D-Printed Scaffold Model to Investigate Embryo Implantation Mechanisms

Co-culture Model Using Two Types of Adherent Cell Lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

Author Spotlight: In Vitro Endometrial Models to Study Epithelial-Stroma and Embryo Interactions

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications ...