This research was supported by a National Leading Research Laboratory Program (Grant NRF-2013R1A2A1A05006378) through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning. The authors also acknowledge a KAIST Systems Healthcare Program.

&#160; &#160;1. Preparation of the Micropatterned Molds and Hydrogels
<ol>
	<li>Produce the desired micro-scale patterns using SU-8 photoresist on the surface of a silicon wafer via a standard two-step photolithography technique15,17 for casting PDMS molds. The example shown uses a liver lobule-like mesh pattern (Figure 1).</li>
	<li>Weigh out PDMS and a curing agent solution with a ratio of 1:5 (i.e., 12.5 g of PDMS and 2.5 g of curing agent).</li>
	<li>Mix the 15 g of the solution thoroughly, degas the bubbles in a vacuum chamber, and then spread the mixed solution onto a micropatterned surface of the silicon wafer evenly within a foil casting dish.</li>
	<li>Place the silicon wafer onto a 65 &#176;C heated plate for 90 min on a flat surface to cure the PDMS.</li>
	<li>Remove the cured PDMS from the casting dish and the silicon wafer.</li>
	<li>Cut the edges of the PDMS and place it onto a 100-mm-diameter petri dish with the micropatterned side up.</li>
	<li>Wash the micropatterned cured PDMS on the petri dish using 70% ethanol and distilled water for primary sterilization. Then, dry them completely for 10 min in an oven at 65 &#176;C.</li>
	<li>Dissolve and mix O/N&#160;3 g of a powdered nonionic surfactant in 100 ml of distilled water, creating a 3% (w/v) coating solution.</li>
	<li>Place the micropatterned PDMS molds in a plasma cleaner and clean them for 1 min (85 W, 0.73 mbar) to create a hydrophilic surface, to facilitate the addition of aqueous liquids. Then, coat the surface of the PDMS with the 100 ml surfactant solution for at least&#160;3 hr (orO/N) using a laboratory rocker.</li>
	<li>Wash the surfactant solution from the PDMS molds and dry them completely in an oven at 65 &#176;C for 10 min. Then, sterilize each micropatterned mold by exposure to ultraviolet (UV) radiation over 30 min.</li>
</ol>
2. Prepare the Cell Suspension in a Hydrogel Precursor
<ol>
	<li>To prepare hydrogel precursor, dissolve 0.1 g of sodium alginate powder in 10 ml of phosphate buffered saline (PBS), creating a 1% (w/v) alginate precursor. To dissolve the powder completely, incubate and mix them O/N.</li>
	<li>Filter the solution through a 0.22&#160;&#181;m filter connected to a 1&#160;ml syringe.</li>
	<li>Culture the HepG2 cells in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin on a conventional tissue culture dish until 70% - 80% confluence in a 5% CO2 humidified incubator at 37 &#176;C.</li>
	<li>Wash the cells once using PBS, and then trypsinize them by adding 1 ml of 0.05% trypsin-EDTA for 4 min in a 5% CO2 humidified incubator at 37 &#176;C. Centrifuge the cells harvested from the culture dish at 250 x g for 3 min and resuspend using 1 ml of PBS following removal of the supernatant.</li>
	<li>Count the number of single distributed cells in PBS using an automated cell counter.</li>
	<li>Following centrifugation at 250 x g for 3 min and PBS removal, add 1 ml of the 1% (w/v) of sodium alginate solution to the remaining cell pellet and mix them gently using pipetting. The final seeding density of the cells should be 5 x 106&#160;- 107 cells/ml. Incubate the cell/hydrogel suspension in a 5% CO2 humidified incubator at 37 &#176;C.</li>
</ol>
3. Loading and Cross-linking of Cell/Hydrogel Suspension
<ol>
	<li>Dissolve 1.47 g of calcium chloride dehydrate in 100 ml of ddH2O to produce a cross-linking reagent (i.e., 100 mM CaCl2&#183;2H2O in ddH2O).</li>
	<li>Rinse out the interior of a humidifier with ultrasonic transducer using 70% ethanol, and fill it with 200 ml of cross-linking reagent. The humidifier is 110 mm wide, 300 mm long and 170 mm deep (i.e., 110 mm x&#160;300 mm x 170 mm (W x H x D)).</li>
	<li>Place the micropatterned PDMS molds in a plasma cleaner and clean them for 1 min at 85 W to create a hydrophilic surface.</li>
	<li>Steadily load 7.2 &#181;l of the well-mixed cell/hydrogel suspension at the edge of the micropattern in the mold. The example shown uses a liver lobule-like mesh pattern (Figure 1). The volume of the suspension depends on the topographic pattern.</li>
	<li>To achieve gelation of the cell/hydrogel suspension, turn on the humidifier and verify that the humidifier produces a mist of the cross-linking reagent. Spray the cross-linking reagent onto the hydrogel precursor for 5 min, covering the topographic surface of the PDMS molds within a range of 5 cm. 
	Note: Distances longer and shorter than 5 cm could cause incomplete and uneven gelation, respectively. Ensure that the humidifier has a spraying rate of 250 ml/hr, producing 20 ml of mist of the cross-linking reagent in 5 min.</li>
	<li>Following the cross-linking process, turn off the humidifier and fill the PDMS molds with PBS.</li>
</ol>
4. Handling of Single Modular Hydrogel Sheets
<ol>
	<li>Detach each hardened hydrogel sheet from the micropatterned molds via pipetting PBS gently around the hydrogel sheet using a 200&#160;&#181;l pipette tip.</li>
	<li>Pick up each floating hydrogel sheet using an end-cut 1,000&#160;&#181;l pipette tip end. Each liver lobule-like mesh hydrogel sheet has dimensions of 8 mm x 8.7 mm, and be 100 - 200 &#181;m thick.</li>
	<li>Transfer a single layer of the hydrogel sheet into 1 ml of DMEM in a 12-well plate, and culture the cells in vitro in a floating manner using the hydrogel construct as a unit component in the 12-well plate over a week in a 5% CO2 humidified incubator at 37 &#176;C. Exchange the culture medium every other day.</li>
</ol>
5. Assembly of Multi-layered Hydrogel Sheets
<ol>
	<li>Repeat steps 1.1 to 1.5 to produce a PDMS frame with dimensions of 18 mm x 18&#160;mm x 4 mm (W x H x D), and which contains 170-&#181;m-high pillar structures at the lower surface. Use 42 g of the mixture of PDMS and curing agent, with the same ratio as in step 1.2. Place a specialized polycarbonate mold on the silicon wafer for the PDMS frame to create an interior frame with dimensions of 8 mm x 9 mm x 2 mm (W x H x D).</li>
	<li>Sterilize the PDMS frame and 180-&#181;m-pore nylon filter papers&#160;submerged in distilled water and tweezers in an autoclave for 15 min at 121 &#176;C.</li>
	<li>Place the sterilized PDMS frame onto a quarter of a piece of nylon filter paper (with a diameter of 5 cm) in a 60-mm diameter&#160;petri dish.</li>
	<li>Transfer a modular hydrogel sheet into the interior of the PDMS frame using an end-cut 1,000&#160;&#181;l pipette tip. The hydrogel sheets used for assembly should be cultured for at least a day after they were fabricated.</li>
	<li>Align the edge of each modular hydrogel sheet with the PDMS frame using an empty 200&#160;&#181;l pipette tip.</li>
	<li>Repeat steps 5.4 and 5.5 using modular hydrogel sheets to form a stack of 4 - 6 layers.</li>
	<li>Remove the culture medium by flowing it out through the pillar structures at the bottom of the PDMS frame. Then add 2 &#181;l of alginate solution (2% w/v) at a corner of the multi-layer construct.</li>
	<li>Spray a mist of the cross-linking reagent for 30 sec onto the multi-layer construct to attach the edges of each layer with those of another. Use 2 ml of mist of the cross-linking reagent (at a spraying rate of 250 ml/hr).</li>
	<li>Rinse the multi-layered construct gently with 400 &#181;l DMEM and remove the PDMS frame using tweezers.</li>
	<li>Detach the multi-layered construct from the filter paper by gently wiping with a cell lifter following the addition of 4 ml of DMEM.</li>
	<li>Transfer the multi-layered construct to a 6-well plate containing 3 ml of DMEM using filter paper, and culture the cells in vitro in a floating manner, with the hydrogel construct as a multi-scale cellular scaffold in the 6-well plate over a week in a 5% CO2 humidified incubator at 37 &#176;C. Exchange the culture medium every other day.</li>
</ol>

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The authors have nothing to disclose.

This protocol provides a simple method of fabricating modular hydrogel sheets, and assembling them to form 3D cellular scaffolds.
To construct clear-cut patterned alginate structures in a short time, we should identify a cross-linking process that can create sufficiently rigid structures to maintain the complex micropatterns from the mold, as well as maintain cell viability and metabolism. We have developed a cross-linking process, including a sol&#8211;gel transition, to spray a cross-linking reagent using a humidifier in the form of a mist. Without this cross-linking process, the micro-scale patterns could not be generated in the form of a thin hydrogel construct with the limited diffusive depth (100 - 200 &#181;m), which has high diffusive permeability to metabolites and nutrients. Furthermore, compared with existing diffusion-based methods, which require 30 - 60 min,18&#8722;20 the cross-linking time was relatively short (3 - 5 min).
Moreover, it was also challenging to manipulate the thin hydrogel sheets, which were mechanically stable only in a liquid medium. As shown in Figure 1, the thin hydrogel structure was simply and stably handled and manipulated using an end-cut conventional tip for transferal between different culturing conditions. Using generation and manipulation techniques, we could provide not only a single-layer sheet in a floating manner for potential applications in drug assays but also stacked multi-layer structures, with potential applications as scaffolds for tissue-like constructs.
Multi-layered cellular architectures can be generated by stacking the modular single-layer hydrogel sheets (Figure 3A). Each modular hydrogel sheet may contain differing cell types, and may be exposed to different culture conditions. These multi-layer constructs may then be selectively assembled. However, simply stacking hydrogel sheets may lead to cell necrosis inside the assembled structure when the total depth exceeds the limited diffusive depth (Figure 3C). To overcome this problem, it would be necessary to align micromesh patterns, such as liver lobule-like meshes, when the multi-layer hydrogel sheets are assembled. We therefore require a PDMS frame with the same interior pattern as the border of the hydrogel sheet to align the stacked hydrogel sheets. Thus, aligned hole-array micropatterns in the assembled mesh hydrogel sheets could be used to form expanded pipeline structures, facilitating more efficient nutrient transport for improved cell viability (Figure 3B).
One limitation of this technique is the absence of cell&#8211;matrix interactions in the alginate hydrogel. Alginate cannot provide cell adhesion ligands for in vivo-like cell&#8211;matrix interactions;21 however, it does offer mechanical and geometrical effects as a 3D cell-encapsulating scaffold with cell&#8211;cell interactions. For primary cells and stem cells, alginate could be used following modification with RGD-containing peptides,21 or in combination with collagen or gelatin.14,18 Another limitation is that, once the cell-embedded hydrogel sheets are fabricated with low cell density below 5 x 106 cells/ml, construction of multi-layered structure could cause weak attachments between the hydrogel sheets because only the edge of the structure is fixed by use of additional alginate. However, the layers can be stacked compactly as a single scaffold via drainage structure under the PDMS frame. In addition, higher density of cells in the hydrogel sheets could proliferate on the surface as well as within the layer after one day culture, which would help attachments between layers as shown in Figure 3A.
The hydrogel sheet fabrication and manipulation technique described here may be adapted to the in vitro culture systems and tissue-like cellular models to provide an in vivo-like microenvironment for applications, including cellular assays and engineering artificial organs. This technique could be applied to cell-based drug assays and biological studies that require geometrically different 3D cellular micro- and macroenvironments that incorporate various cells or hydrogel types.

Hidrojeller özellikle biyomalzemeler umut vericidir, ve temel biyoloji, farmakolojik deneyleri ve tıpta önemli olması beklenmektedir. 1 hidrojel tabanlı hücresel yapıları Biofabrication hayvan deneyleri kullanımını azaltmak için ileri sürülmüştür, 2,3, 4 transplante dokuların yerini ve geliştirmek Hücre bazlı deneyler. 5,6 su-ihtiva eden (hidro) viskoelâstik malzemelerin (jeller), hücrelerin geniş bir sayısı kapsüllenmiş ve 3B, hücresel mikro-kontrol etmek için bir iskele yapısı içinde muhafaza edilmesini sağlar. Mikroakışkan ya micropatterning teknolojileri yönlendirmesi ile birlikte, hidrojel yapıları geometrisi kesin hücresel ölçekte kontrol edilebilir. Tarih, parçacıkların dahil hidrojellerin şekiller, çeşitli, 7-9 lifler, 10-12 ve levhalar, 13-15 aşağıdan yukarıya beğenme yapı birimi olarak kullanılmıştırMakro ölçekli çok hücreli mimarilerinin imalat için ağrıyor. Hidrojel-bazlı partiküller ve elyaflar Hem mikroakışkan cihazlar kullanılarak akışkan kontrol grubu ile, mikro ölçekli hücre ortamları gibi uygulamalar için kolaylıkla ve hızlı bir şekilde imal olmuştur. Bununla birlikte, mühendislik dokuların temel birimleri olarak, onları yeniden düzenlemek ve makro ölçekli yapıları olarak hacmini büyütmek için. 16 mikron büyüklüğünde temel modülleri üretmek için daha Makro ölçekli yapıları elde etmek daha zordur karmaşık olacaktır. Hidrojel-bazlı yapıların Levha benzeri birimler basit bir montaj işlemi ile iskeleler hacmini artırmak için de kullanılabilir. Neticesinde, hidrojel levha istiflenmiş katmanları bir hacimsel artış, aynı zamanda 3D uzayda geometrik uzantısı sadece sağlar. Çoklu yumurtlama içine montajı ile birlikte 15 - Daha önce, 13 micropatterned hidrojel tabakaları imal edilmesi için bir yöntem bildirmiştirola- rak hücresel mimarileri. Teknik, çok katmanlı yapıların istifleme işlemi yoluyla karmaşık micropatterning ve hücresel yapıları modüler tasarımı sağlar. Micropatterned edilir istiflenmiş modüler hidrojel levha, imalatı ile, kontrollü bir makro-ölçekte, hücresel mikro-3 boyutlu bir hücre kültür sistemi gerçekleştirilebilir. Bu video protokol insan karaciğer karsinoma hücre hattında (HepG2) göre modüler hidrojel tabakaları oluşturmak için kullanılabilen basit ama güçlü bir üretim yöntemi açıklanmaktadır. Bu tarifnamede, basit olan bu desenli modüler hidrojel tabakaların manipülasyonu ve bir çok-tabakalı yapı halinde asamblajı göstermektedir.

<table border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:111px;">Dow Corning Corporation</td>
			<td style="width:163px;">000000000001064291</td>
			<td style="width:397px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:288px;">Pluronic F-127</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:163px;">P2443</td>
			<td>Powdered nonionic surfactant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Alginic acid sodium salt, low viscosity</td>
			<td style="width:111px;">Alfa Aesar</td>
			<td style="width:163px;">B25266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Calcium chloride dihydrate</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:163px;">C7902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Ultrasonic humidifier</td>
			<td style="width:111px;">MediHeim</td>
			<td style="width:163px;">MH-2800</td>
			<td>Modified equipment, Maximum sprayed rate: 250 mL/h</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:288px;">Nylon net filter hydrofilic, 180 &#956;m</td>
			<td style="width:111px;">EMD Millipore</td>
			<td style="width:163px;">NY8H04700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Polycarbonate mold</td>
			<td style="width:111px;"></td>
			<td style="width:163px;"></td>
			<td>Customized mold for fabrication of a PDMS frame pattern</td>
		</tr>
	</tbody>
</table>

construction of modular hydrogel sheets for micropatterned macro-scaled 3d cellular architecture

Hidrojeller, bir in vivo benzeri üç boyutlu (3D), doku yapısının sağlanması için mikro-akışkan veya micropatterning teknolojileri kullanılarak mikro ölçekte desenli olabilir. Elde edilen 3D hidrojel-bazlı hücresel yapılar gelişmiş biyolojik çalışmalar, farmakolojik tahliller ve organ nakli uygulamaları için hayvan deneylerinde alternatif olarak sunulmuştur. Hidrojel-bazlı partiküller ve elyaflar, kolayca imal edilebilir, ancak, bu doku onarımı için bunları işlemek oldukça zordur. Bu videoda, biz kontrollü hücresel mikroçevresinin ile makro ölçekli 3D hücre kültürü sistemi oluşturmak için kendi montaj ile birlikte, micropatterned aljinat hidrojel sayfaları için bir fabrikasyon yöntem açıklanmaktadır. 200 um ve hassas micropatterns ile - kalsiyum jelleştirme maddesinin bir sis formu, ince hidrojel levha kolayca 100 aralığı içinde bir kalınlığa sahip olarak oluşturulur. Hücreler daha sonra hidrojel yaprak geometrik rehberlik ile kültüre edilebilirduran koşullar. Ayrıca, hidrojel levha hali hazırda bir uç kesim ucu ile bir mikropipet kullanılarak manipüle edilebilir ve desenli bir polidimetilsiloksan (PDMS) kare kullanarak istifleme tarafından çok tabakalı yapılar halinde monte edilebilir. , Basit bir işlem kullanılarak imal edilebilir Bu modüler hidrojel levha, mikro ve makro ve doku yeniden üzerindeki fonksiyonel çalışmalarda da dahil olmak üzere in vitro ilaç deneylerinde ve biyolojik çalışmalar, potansiyel uygulamaya sahiptir.

We have described the fabrication and manipulation of freestanding cellular hydrogel sheets. As shown in Figure 1, we fabricated micropatterned PDMS molds, and cell-containing hydrogel was loaded onto the hydrophilic surface of these molds and cross-linked using a humidifier to generate an aerosolized mist of gelling agent. Following release from the molds, HepG2 cells were cultured in freestanding hydrogel sheets with various patterns (Figure 2). Thus, the thin hydrogel sheets provided a 3D culture environment. Furthermore, modular hydrogel sheets may be assembled by layering the sheets using a micropipette with an end-cut tip, and using a PDMS frame, which enables 3D macro-scale cell culture (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/53475/53475fig1.jpg" /> 
Figure 1. Flow Chart Showing the Fabrication of a Hydrogel Sheet and Multi-layer Hydrogel Sheets. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53475/53475fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53475/53475fig2.jpg" /> 
Figure 2. Freestanding 3D Cell Culture in Various Hydrogel Sheets. The fabrication method of spraying cross-linking reagent enabled precise patterning of alginate hydrogel at the micro-scale. Furthermore, the fabricated hydrogel sheets could be harvested from the mold and manipulated easily in freestanding conditions. HepG2 cells were patterned and cultured in a flat hydrogel sheet (A), hydrogel sheets with hexagonal pillars (B), mesh (C), liver lobule-like mesh (D), an array of holes (E), and multiple microcomb-like microfibers (F). The scale bars are as follows: 500 &#181;m (A, D, F), 100 &#181;m (B, C), and 2 mm (E). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53475/53475fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53475/53475fig3.jpg" /> 
Figure 3. Assembly of Micropatterned Hydrogel Sheets for Macro-scale Cellular Constructs. Various types of hydrogel sheet could be assembled via layer-by-layer stacking with alignment, including the assembly of five hydrogel sheets containing HepG2 cells stained with green fluorescence and NIH3T3 cells stained with red fluorescence (A). An assembly of micropatterned hydrogel sheets with expanded pipeline structure (B) provided higher cell viability than that of non-patterned hydrogel sheets (C) after 7 days. The viability was assessed by staining HepG2 cells with calcein-AM (live cells shown as green) and ethidium homodimer-1 (dead cells shown as red). Scale bars: 500 &#181;m (A) and 200 &#181;m (B, C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53475/53475fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture at JoVE.com

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Micropatterned Makro ölçekli 3D Hücresel Mimarlık Modüler Hidrojel Levhaların İnşaatı

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

construction-modular-hydrogel-sheets-for-micropatterned-macro-scaled

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K Görüntüleme.  Korea Advanced Institute of Science and Technology (KAIST). We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment. 

Video: Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Doku Mühendisliği: Katmanlı Hücre Sheets teslimi için bir Multisellüler 3D İskele İnşaatı

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Biyomühendislik

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

Hücresel Mimics İnşası için Veziküller Hücre-ücretsiz Transkripsiyon ve Çeviri Makine Sarma

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Eklem Kondrosit Kültür ve Kıkırdak Üretimi için 3D hidrojel iskeleleri

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Hücre yüklü hidrojel uyarıcı hücresel davranışları için degrade zorlanma çip

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

Geliştirilmiş 3D hidrojel Neuroinflammation Vitro modelleme için birincil gliyal hücreler kültürleri

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable aljinat/jelatin hidrojel 3D Vitro Model sistemleri hücre küresel oluşumu teşvik

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3D dinamik kültür sistemi ile bir çok katmanlı mezenkimal kök hücre levha inşaatı

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Chemoattractant degradelere Multi-hücresel tepkiler 3D Analizi

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D gözenekli selüloz Nanocomposite hidrojel iskele baskılı

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Jelatin Metacryloyl Hidrojel Bazlı Bioinks 3D Biyobaskı Protokolleri

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...