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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(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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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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Research

JoVE Journal

Biology

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.  ...

Applying Compounds to Citrus Plants Using a Novel 3D-Printed Direct Plant Infusion (DPI) Device

To apply compounds to the plants using the Novel 3D-Printed Direct Plant Infusion, or DPI device, ensure the device is properly fitted to the plants. When assembled properly, the plastisol ring should be flush against the DPI device and the spout should line up with the hole drilled in the tree. Use a syringe or pipette to fill the DPI device with the compound solution of interest.Using a syringe, penetrate the silicone tape and plastisol ring opposite to the DPI device to extract air from the interior channel. Replace any extracted compound back into the DPI device. Add an additional small patch of silicone tape over the hole created by the syringe to reinforce the area and prevent tears.Inspect the apparatus for visible leakages at the attachment point and inspect the device reservoir for a stable liquid level. Cover the open end of the DPI device with a wax sealing film and pull it down to form a seal to reduce the evaporation of the experimental compound. Poke a single a hole in the wax film with a syringe tip to prevent the development of a vacuum, and subsequently refill the device.Check the apparatus daily to ensure proper alignment of the components, and top off the liquid using a syringe to avoid the reservoir running dry til the desired amount is delivered. When two milliliters of two millimolar CFDA dye was introduced to the plant using the DPI device, a fluorescent signal was detected in the vasculature of the treated plant, but was absent in the controlled plant treated with 20%DMSO in water. This signal was observed within 24 hours of treatment, evenly throughout the dissected plant tissue types.Treatment of streptomycin on Candidatus Liberibacter Asiaticus or CLass positive plants showed a reduction in the mean bacterial titer to 28 days after treatment compared to the control. The treated plants showed a lower mean CLas titer over all combined time points. The streptomycin treated plants as compared to the controls showed an occasional increase in new healthy flush growth after 60 days.Imidacloprid treatment on plants significantly reduced nymph emergence at the highest Imidacloprid concentration compared to the corresponding egg count. This was visually apparent in the reduction in nymph honeydew production.

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 ...

Generation of Rat Intestinal 2D Monolayers from 3D Organoids

To coat the plate with EME, dilute EME 1 to 20 in cold Advanced DMEM+For coating with collagen, dilute five milligrams per milliliter of collagen I in Advanced DMEM+to 100 micrograms per milliliter. Coat with the plate with 200 microliters of diluted EME or collagen to cover the well surface entirely and incubate for one to two hours at 37 degrees Celsius. To generate monolayers, aspirate the media from a 35 millimeter plate containing organoids.Add one milliliter of PBS and disrupt the EME in the wells with a P1000 tip, pipetting up and down approximately 20 times to loosen all the EME. Transfer the contents to a 15 milliliter conical tube. Add one milliliter of PBS to the plate to collect additional organoids and transfer them to the same conical tube.Centrifuge the sample at 350 G for two minutes and remove the supernatant, including any EME residue. Then, add one milliliter of trypsin solution to the organoid pellet and incubate at 37 degrees Celsius for two minutes. Pipette up and down 10 times using a P1000 tip and add two milliliters of Advanced DMEM+to neutralize trypsin.Centrifuge at 350 G for five minutes and aspirate the supernant before resuspending the pellet in 4.8 milliliters of rIOM2D. Remove excess EME or collagen in Advanced DMEM+from the wells. Then, add 200 microliters of organoids in rIOM2D and 10 micromolar Y27632 to each pre-coated well.After 4 to 16 hours, collect the media and centrifuge at 1, 000 G for one minute. Transfer the supernatant into a new 15 milliliter conical tube and discard the pellet. Wash each well with 300 microliters of PBS before adding 200 microliters of the centrifuged rIOM2D to each well.Change the rIOM2D every two to three days, suspending the use of Y27632.2D monolayers plated on the EME readily reformed into small spheroids when EME was added back to the top of the cells. In contrast, a collagen I substrate was insufficient to reform 3D structures.

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 ...

Installing the Live Imaging Insert on an Inverted Microscope Stage

To begin installing the 3D printed live imaging insert on the inverted microscope stage, carefully place the insert into the large microscope stage groove. Using screws, secure each corner of the insert. Slide the heat plate into the side opening of the insert in a plug side down orientation, so the plate lays atop the lower insert grooves with the plug port underneath the stage.Align the grooved circular opening in the insert with a 40 x silicone oil immersion objective and apply silicone oil to the center of the objective. Then using a syringe, apply a small amount of vacuum grease along the grooved circular opening. Lay the cover slip disk on top of the opening to seal it onto the insert.Finally, raise the 40 x objective until it barely touches the bottom of the glass cover slip.