This work was supported by the Foundation for Research Support of the State of Rio de Janeiro (FAPERJ, Brazil), the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil), and the Brazilian National Council for Scientific and Technological Development (CNPq, Brazil). We thank Bioedtech for providing the stamp-like devices used in this study and Professor Bartira Bergmann from the Immunopharmacology Laboratory for the use of their cell culture facilities.

The L929 cell line, mouse fibroblasts, was used for the present study. The stamp-like 3D printed biodevice was obtained from a commercial source (see Table of Materials). Good cell culture practice and sterile techniques were followed throughout the protocol. The fabricated device was sterilized by wiping it with 70% alcohol and exposing it to UV light for 15 min. The cell culture media and solutions were warmed to 37 &#176;C before contacting with the cells or tissue spheroids. A schematic representation of the protocol is shown in Figure 1.
1. Preparation of non-adherent molds from the stamp-like device
<ol>
	<li>Prepare 2% (w/v) agarose gel following the steps below.
	<ol>
		<li>Dilute the agarose powder in phosphate-buffered saline (1x PBS) and homogenize the resulting suspension with circular movements. 
		NOTE: This solution can be placed in a glass bottle. In this step, the agarose solution will not be translucent.</li>
		<li>Place the glass bottle into the microwave and set it for 30 s. Every 5 s, stop the microwave, remove the glass bottle, and manually homogenize the solution with circular movements. The heating process needs to be performed until the solution reaches a liquid-limpid state. 
		NOTE: A hot plate can also be used as a microwave alternative. After the heating process, the solution must be translucent/limpid.</li>
		<li>Add 1 mL of the agarose solution to each well of a 6-well plate planned for the experiment.</li>
		<li>Wait for ~15 min or until the agarose solidifies. 
		NOTE: A cooling plate can be used to decrease the solidification time.</li>
		<li>Add 1-2 mL of the agarose solution and gently insert the device above the liquid agarose. 
		NOTE: The placement of the device must be done carefully to prevent air bubbles in the agarose-device interface.</li>
		<li>Wait for ~30 min or until the agarose solidifies. 
		NOTE: A cooling plate can be used to decrease the solidification time.</li>
		<li>Gently remove the device from the agarose. 
		NOTE: The removal is critical. One should remove it carefully to maintain the agarose features intact; otherwise, the agarose can be disrupted.</li>
		<li>Add 2 mL of DMEM media, wait for 10 min, discard the media, and replace it with fresh DMEM. Repeat this three times to wash the well properly.</li>
		<li>Add 2 mL of DMEM and place the 6-well plate in an incubator (at 37 &#176;C in 5% CO2 and 80% humidity) until the cell seeding (Figure 2A-F, Supplementary Video 1).</li>
	</ol>
	</li>
</ol>
2. Generation of tissue spheroids
NOTE: Different cell lineages have different adhesion properties. Hence, using this methodology, some types of cells may not form the tissue spheroids properly.
<ol>
	<li>Grow the cells following traditional monolayer culture (i.e., grow the cells in cell culture flasks using DMEM with low glucose supplemented with 10% fetal bovine serum (FBS), 100 &#181;g/mL penicillin, and 100 &#181;g/mL streptomycin) (see Table of Materials). Maintain the cells at 37 &#176;C in a 5% CO2 incubator, and monitor until they reach 80% of confluence.</li>
	<li>After reaching the desirable confluence, wash the cells with 1x PBS. 
	NOTE: It is recommended to use 5 mL for 25 cm2 flasks , 10 mL for 75 cm2 flasks, and 15 mL for 150 cm2 flasks.</li>
	<li>Add the dissociation enzyme and incubate the cells for 2-5 min at 37 &#176;C in 5% CO2 and 80% humidity. 
	NOTE: The present study used 0.125% trypsin with 0.78 mM ethylenediamine tetraacetic acid (EDTA) as the dissociation enzyme (see Table of Materials).</li>
	<li>Observe the detachment of the cells from the cell culture flasks and add a growth medium supplemented with FBS to neutralize the cell dissociation enzyme. 
	NOTE: For the present study, DMEM with low glucose (see Table of Materials) was used supplemented with 10% FBS.</li>
	<li>Centrifuge the cell suspension at 400 x g for 5 min at room temperature. Then, count the cells manually.</li>
	<li>Take 50 x 105 cells per tube and add 5 mL of 1x PBS. 
	NOTE: The number of cells seeded influences the final tissue spheroid diameter. Hence, one can increase the cell number to generate tissue spheroids with larger diameters.</li>
	<li>Centrifuge the cell suspension at 400 x g for 5 min at room temperature.</li>
	<li>Remove the supernatant using a pipette, add 1 mL of the cell culture medium, and homogenize the solution. 
	NOTE: In the present study, a complete culture medium of DMEM with low glucose, supplemented with 10% FBS, 100 &#181;g/mL penicillin, and 100 &#181;g/mL streptomycin, was used.</li>
	<li>Remove 2 mL of medium from the 6-well plate (step 1.1.9) and add 1 mL of the cell suspension to the center of the agarose mold formed by the 3D-printed biodevice (step 1.1.7). Wait until the cells sediment in the micro resections (~20-30 min) and carefully add 1 mL of cell culture medium into the well. 
	NOTE: One needs to be extra careful in this step. It is recommended to gently add the medium, place the pipette tip close to the well wall, and dispense in small amounts.</li>
	<li>Place the 6-well plate in the incubator (at 37 &#176;C in 5% CO2 and 80% humidity) for the tissue spheroids to form (approximately 24-48 h, depending on the cell type) (Figure 3). 
	NOTE: Different cell types (e.g., cancer cells, primary cells) have different self-assembly kinetics11.</li>
</ol>

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The 3D printed stamp-like devices were offered by the startup Bioedtech, in which Jana&#237;na Dernovsek is the cofounder and innovation director. The authors declare no competing financial interests.

The present protocol describes a simple, fast, and inexpensive method for the large-scale production of tissue spheroids. A stamp-like 3D printed device was used as a master mold, which generated up to 4,716 spheroids per 6-well plate. It has been shown that cells cultivated as spheroids have more realistic biological responses that closely resemble the in vivo environment1. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models13,14. Hence, developing new methodologies to achieve large-scale production is pivotal to leveraging their application.
To successfully achieve the generation of tissue spheroids using this methodology, critical steps such as inserting the device into the liquid agarose need to be performed with caution to avoid air bubbles. The presence of air bubbles might impair the correct molding of the micro resections. Accordingly, removing the device before total solidification of the agarose may result in the rupture of the agarose mold. These steps are essential to produce agarose molds suitable for tissue spheroid formation. The shape and size variability for the generated batch of spheroids needs to be minimal. Therefore, one of the deterministic points is the slow addition of the culture medium after the cells are seeded in the agarose mold. This requires gentle dispensing of the liquid to not disturb the cells that have already entered the micro resections. Otherwise, some micro resections could have augmented concentrations of cells, which will lead to heterogeneity in size among the spheroids.
In addition to being simple, fast, and inexpensive, this protocol is also flexible (i.e., some modifications in it are possible to adjust to the experiment&#39;s particularities). Alternatively to agarose, one can use polydimethylsiloxane (PDMS) to form the molds. PDMS is an elastomer that is broadly used to fabricate microfluidic chips15. It has properties such as biocompatibility, optical transparency, being chemically inert, thermal stability, and gas permeability15. Different from agarose, molds made with PDMS are more resistant and can also be reused with preservation of the micro resection geometry.
Currently, the search for an alternative serum-free cell culture medium has increased worldwide16,17. Although FBS is a traditional supplement for cell culture, its use has ethical issues and possible batch variability. Nonetheless, considering the clinical application of spheroids, the risks of immune reactions and disease transmission impair the use of FBS as a medium supplement16. Thus, one can use a serum/xeno-free cell culture medium to form the spheroids following the present methodology. For instance, studies have used a special 3D culture medium composed of human albumin, ascorbic acid, insulin, transferrin, and selenium to form human mesenchymal/stromal stem cell spheroids4,8.
The successful formation of spheroids can be determined by their compact spheroidal shape. To date, some studies have reported the impact that cell adhesion mediators (e.g., E-cadherin, integrins), as well as cytoskeleton microtubules and actin filaments, have on cell self-assembly and further compaction into spheroids18,19. Therefore, cells that present disbalance in expressing these components might not be able to compact and form the spheroids. Hence, spheroid formation differs according to the cell line; thus, some types of cells might not be suitable options for the methodology described herein.
In the present protocol, a 3D printed stamp-like device, composed of cylindrical micropins, was used to mold a non-adherent hydrogel (e.g., agarose) to form an array of uniform microstructured resections. It is also possible to observe (i.e., using imaging) the cell&#39;s aggregation, self-assembly, and compaction in real time. Moreover, one has minimal labor to add/exchange medium and add drugs or other compounds. In addition, one can withdraw the spheroids for post-analysis, such as electron microscopy, cytometry, and histology, by merely applying a directed jet using a pipette loaded with medium or PBS. Several methodologies have been reported to generate spheroids, such as hanging drop, coated u-shaped bottom wells, microfluidics devices, and using a polymeric matrix. Nonetheless, they are usually complex, time-consuming, labor-intensive, expensive, or may generate heterogeneous spheroids 9,10. Although the large-scale production of spheroids was achieved using the hanging drop methodology20, it is still difficult to follow the spheroid formation process and to perform medium exchange or the addition of drugs/compounds10.
Micromolded non-adhesive hydrogels have been used as a strategy to form spheroids and also molds to guide the formation of larger microtissues10. However, the master molds used are mainly silicon, are unsuitable for long-term use, and might be expensive21. This study employed stereolithography (SLA) for the 3D printing technology to generate highly uniform master molds. SLA generates the prototype with speed, cost-effectiveness, flexibility, and precision22. Additionally, its advantages for customized manufacturing have been used in several fields, including tissue engineering and biomedicine in general22.
It is known that spheroid cell cultures are physiologically relevant in vitro study models that can closely resemble human features. Hence, they have been used as a model to study the onset of diseases and toxicology and are already commercialized as a bioproduct for regenerative medicine23,24,25. Accordingly, they are used as the raw material for forefront technologies such as 3D bioprinting3,26 and organs-on-chips24,27, continuously showing how significant spheroids cultures are and the importance of developing new strategies to generate them on a large scale.

Tissue spheroids are 3D micro-tissues formed by cell suspensions that undergo self-assembly without external forces1. These spheroids have been widely used in biofabrication protocols due to their resemblance with key features of the human physiological system2,3. Tissue spheroids provide more similar metabolism, cytoskeleton dynamics, cell viability, and metabolic and secretion activity than traditional monolayer cell culture1. Due to their fusion capability, they can also be used as building blocks (e.g., bioprinting protocols) to form complex tissue-engineered constructs with enhanced biological relevance4,5.
Due to their biological relevance, tissue spheroids have been used as a biotechnological tool for protocols ranging across tissue engineering, drug development, disease modeling, and nanotoxicological assessment, reducing time, space costs, and animal testing3,6,7,8. Nonetheless, to fully explore and leverage the potential of tissue spheroids, reliable and reproducible methods aiming at their large-scale production are highly necessary, and these remain an ongoing challenge.
Several methodologies produce spheroids, such as hanging drop, coated u-shaped bottom wells, microfluidics, and using a polymeric matrix9,10. Although these methodologies paved the way within the spheroid production market, they are still complex, time-consuming, labor-intensive, or expensive10.
The present protocol reports the large-scale production of homogeneous tissue spheroids using a low-cost and time-effective methodology. We have developed a 3D printed stamp-like device to generate up to 4,716 spheroids per 6-well plate. Moreover, the stamp-like device can be tailored to produce more spheroids per well, suitable for different cell culture plates. It is easily sterilizable and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to translate their use to the clinics, contributing to multiple areas of industry such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6 well plate</td>
			<td>Merck</td>
			<td>CLS3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Promega</td>
			<td>V3121</td>
			<td></td>
		</tr>
		<tr>
			<td>Biodevice&#160;</td>
			<td>Bioedtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet</td>
			<td>ThermoFisher&#160;</td>
			<td>51029701</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugue</td>
			<td>ThermoFisher&#160;</td>
			<td>75004031</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 50 mL centrifuge tubes</td>
			<td>Merck</td>
			<td>CLS430829-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning cell culture flasks surface area 75 cm2</td>
			<td>Merck</td>
			<td>CLS430641</td>
			<td></td>
		</tr>
		<tr>
			<td>Draft Resin&#160;</td>
			<td>FormLabs</td>
			<td>FLDRBL01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Modified Eagle&#8242;s Medium - low glucose</td>
			<td>Merck</td>
			<td>D6046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher&#160;</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Form 2</td>
			<td>FormLabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>ThermoFisher&#160;</td>
			<td>51033782</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell lines</td>
			<td>Stablished in the lab&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin and Streptomycin (PS)</td>
			<td>ThermoFisher&#160;</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Merck</td>
			<td>806552</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin with EDTA</td>
			<td>Merck</td>
			<td>T3924</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of tissue spheroids via a 3d printed stamp-like device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of homogeneous micro resections using the 3D printed stamp-like device 
The 3D printed stamp-like device was successfully manufactured by the stereolithography method12 using a photocurable resin (Figure 2A). The final device was composed of cylindrical micropins with a height of 1.3 mm and a width of 650 &#181;m (Figure 2A). Its use as a master mold to produce non-adherent micro resections was achieved by maintaining the geometry (Figure 2B-F and Supplementary Video 1). The device was simple to use, easy to sterilize, and could be reused in the long term. Moreover, it is also tunable for different sizes of well plates (i.e., 6 wells, 12 wells, 24 wells, 96 wells) and Petri dishes (i.e., 30 mm, 50 mm, 90 mm, 150 mm). Here, we show data regarding the device for the 6-well plate. It generated 750 homogeneous micro resections per well or 4,716 per 6 well-plate (Figure 2C-D). Drawbacks such as disruption of the non-adherent mold and deformation of the micro resection geometry can occur if the device is withdrawn early (Figure 2G).
Large-scale production of tissue spheroids 
The cells were seeded onto the non-adherent agarose molds, sedimented, and approximately 24 h later formed the tissue spheroids. The large-scale production of the spheroids was achieved by maintaining their shape, size (123 &#181;m &#177; 3 &#181;m), and viability (Figure 3A-D). This methodology supported the spheroids culture for months (data not shown).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63814/63814fig01.jpg" /> 
Figure 1: Schematic representation of tissue spheroid generation using a stamp-like 3D printed biodevice. The biodevice, composed of cylindrical micropins, molds a non-adherent hydrogel (e.g., agarose) to form an array of uniform micro resections. After the solidification of the agarose and the incubation time with the cell culture medium (to acclimatize the new non-adherent molds), the cell suspension is seeded on the molds, and the cells aggregate, self-assemble, and compact into a spherical shape. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63814/63814fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63814/63814fig02.jpg" /> 
Figure 2: The 3D printed stamp-like device used as a master mold to form non-adherent micromolded agarose. (A) The 3D printed stamp-like device. (B) The insertion of the device into the liquid agarose to form the micro resections. (C-E) After the removal of the device, homogeneous resections are formed. (E) Scale bar = 200 &#181;m. (F) Micromolded agarose after incubation with cell culture medium (pink color: cell medium). (G) Removing the device from the agarose when it is not completely solidified leads to its disruption (yellow line with arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63814/63814fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63814/63814fig03.jpg" /> 
Figure 3: Cell seeding and spheroid formation. Phase-contrast microscopy (A) 0 h after the cells seeding in the micro resections (dots within the resections). (B-C) Spheroids formed approximately 24 h after the seeding. Scale bar = 100 &#181;m. (D) Graphic representation of the spheroids&#39; diameter, n = 4. The spheroids formed are homogeneous in shape and size. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63814/63814fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1: Micromolded agarose after removal of the stamp-like device. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63814/Supplementary video 1.wmv" target="_blank">Please click here to download this Video.</a>

Read this Scientific Article Protocol about Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device at JoVE.com

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

generation-of-tissue-spheroids-via-a-3d-printed-stamp-like-device

Nanyang Technological University

Research

JoVE Journal

Biochemistry

1.6K Views.  Federal University of Rio de Janeiro (UFRJ). The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Video: Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various surfaces and interfaces. This non-invasive optical technique can provide the local molecular-level information with monolayer or submonolayer sensitivity. We here are providing experimental methodology on how to selectively detect the buried interface for both macromolecules and biomacromolecules. With this in mind, interfacial secondary structures of silk fibroin and water structures around model short-chain oligonucleotide duplex are discussed. The former shows a chain-chain overlap or spatial confinement effect and the latter shows a protection function against the Ca2+ ions resulting from the chiral spine superstructure of water.

Being comprehensively utilized, sum frequency generation (SFG) vibrational spectroscopy can help to reveal chain conformational order and secondary structural change happening at polymer and biomacromolecule interfaces.

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

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Chapters in this video

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder