eoe sub articles

EoE Sub Articles

1. Synthesis of gelatin methacryloyl (GelMA)
<ol>
	<li>Weigh 10 g of gelatin obtained from porcine skin and dissolve in 100 mL of PBS by letting the solution stir on a heating plate at 240 rpm and 50 &#176;C until complete dissolution.</li>
	<li>Under a hood, add 2 mL of methacrylic anhydride (MA) for a low degree of functionalization, and let the gelatin emulsion stir at 240 rpm and 50 &#176;C for 2 h. 
	CAUTION: MA hazard statement: H302 + H332 (harmful if swallowed or inhaled), H311 (toxic in contact with skin), 314 (causes severe skin burns and eye damage), 315 (causes skin irritation), H317 (may cause an allergic skin reaction), H318 (causes serious eye damage), 331 (toxic if inhaled), H332 (harmful if inhaled), H335 (may cause respiratory irritation). Handling guidelines: P261 (avoid breathing dust/fume/gas/mist/vapours/spray), P305 + P351 + P338 + P310 (IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. Immediately call a POISON CENTER/doctor), P301 + P312 + P330 (IF SWALLOWED: Call a POISON CENTER/doctor if you feel unwell. Rinse mouth). 
	NOTE: Add MA very slowly, drop by drop.</li>
	<li>Dilute the gelatin-MA solution in 100 mL of preheated PBS (50 &#176;C) to obtain 200 mL of final volume and let the solution stir at 240 rpm and 50 &#176;C for 10 min.</li>
	<li>Cut ~20 cm of dialysis membrane (molecular cutoff 12-14 kDa) and soak it in deionized water until it is soft. 
	NOTE: Fill the membranes with deionized water to ensure no holes or defects.</li>
	<li>Using a funnel, transfer the gelatin-MA solution to the membranes. 
	NOTE: Close both sides leaving extra space inside to allow mixture.</li>
	<li>Place the membranes containing the gelatin-MA solution in a container with 2 L of distilled water for dialysis, and let them stir at 40 &#176;C for 5 days (500 rpm). 
	NOTE: Cover the container to avoid water evaporation.</li>
	<li>Change the distilled water two times a day. Each time, flip the membranes upside down for homogenization.</li>
	<li>On the fifth day, mix 200 mL of preheated ultrapure water (40 &#176;C) with the dialyzed gelatin-MA and stir for 15 min at 40 &#176;C.</li>
	<li>Transfer the gelatin-MA solution to 50 mL conical tubes up to 25 mL and let the tubes remain at -80 &#176;C for 2 days. 
	&#8203;NOTE: Store the tubes horizontally to facilitate lyophilization.</li>
	<li>Lyophilize the frozen solutions for 3-5 days and store the lyophilized GelMA protected from humidity.</li>
</ol>
2. Bioink preparation
NOTE: In order to obtain 1 mL of bioink, it is recommended to fabricate at least 3 mL of biomaterial solution, as there may be losses during filtration.
<ol>
	<li>Preparation of fibrinogen solution&#8203;
	<ol>
		<li>Prepare saline solution (NaCl 0.9%) in deionized water, and dissolve 10 mg of fibrinogen from bovine plasma in 1 mL of the saline solution to obtain a concentration of 10 mg/mL. 
		NOTE: As fibrinogen adsorbs to glass, do not use glass flasks to prepare the fibrinogen solution.</li>
		<li>Leave the solution under agitation at 37 &#176;C until complete dissolution of fibrinogen. 
		&#8203;NOTE: For fibrinogen dissolution, use a rotary system placed inside an oven at 37 &#176;C. Magnetic agitation (180 rpm) of fibrinogen on a hot plate at 37 &#176;C is also suitable. Under this condition, 10 mg/mL fibrinogen takes approximately 40 min to dissolve.</li>
	</ol>
	</li>
	<li>Preparation of gelatin/GelMA solution
	<ol>
		<li>Weigh 0.12 g of gelatin and add it to 1.9 mL of preheated PBS (40 &#176;C) to obtain a final concentration of 4% (w/v) gelatin. Vortex to facilitate dissolution.</li>
		<li>Keep the emulsion at 40 &#176;C until complete dissolution.</li>
		<li>Weigh 0.06 g of lyophilized GelMA and transfer it to the gelatin solution to obtain a final concentration of 2% (w/v) GelMA. Vortex to facilitate dissolution.</li>
		<li>Keep the solution at 40 &#176;C until complete dissolution.</li>
	</ol>
	</li>
</ol>
3. Preparation of astrocytes-laden gelatin/GelMA/fibrinogen bioink
<ol>
	<li>Pipette 0.9 mL of the 10 mg/mL fibrinogen solution and transfer to the gelatin/GelMA solution to obtain a final concentration of 3 mg/mL of fibrinogen.</li>
	<li>Weigh 0.015 g of photoinitiator (PI) and transfer to the gelatin/GelMA/fibrinogen solution to obtain a final concentration of 0.5% (w/v) PI. Mix the solution by flipping the tube up and down and keep it at 40 &#176;C protected from light to avoid PI degradation. 
	NOTE: Stock the bioink at 4 &#176;C for a maximum 24 h.</li>
	<li>Under the laminar flow, filter the solution using a 0.2 &#181;m filter into a sterile 15 mL conical tube. 
	NOTE: The biomaterial solution should be at 37-40 &#176;C to allow filtration.</li>
	<li>Transfer 980 &#181;L of the biomaterial solution to a 15 mL conical tube.</li>
	<li>Dilute laminin in saline solution to obtain a stock solution of 100 &#181;g/mL.</li>
	<li>Pipette 20 &#181;L of laminin and transfer to the tube containing the bioink to obtain a final concentration of 2 &#181;g/mL laminin.</li>
	<li>Mix gently by pipetting up and down, avoiding bubbles. If any bubbles persist, centrifuge the conical tube at 200 x g for 2 min. Keep the bioink at 37 &#176;C until it is mixed with the cells.</li>
	<li>Trypsinize primary astrocytes with 0.05% trypsin for 5 min. 
	NOTE: Use astrocytes from passages 1 to 3.</li>
	<li>Neutralize the trypsin activity with FBS at a ratio of 1:1 and transfer the cells to a 15 mL conical tube. Centrifuge it at 200 x g for 5 min.</li>
	<li>Count the cells and transfer 1 x 106 cells to a different conical tube. Centrifuge it at 200 x g for 5 min.</li>
	<li>Remove the supernatant leaving a small volume (~200 &#181;L) to suspend the cell pellet, by gently tapping the bottom of the conical tube.</li>
	<li>Transfer 1 mL of gelatin/GelMA/fibrinogen solution to the tube containing the cells and gently pipette up and down to homogenize, obtaining a final concentration of 1 x 106 cells/mL.</li>
</ol>
3. Preparation of the crosslinker solution
<ol>
	<li>Thrombin reconstitution&#8203;
	<ol>
		<li>Prepare a stock solution of thrombin 100 U/mL in sterile deionized water with 0.1% (w/v) bovine serum albumin (BSA) in a 15 mL conical tube. Stock in microtubes at -20 &#176;C. 
		&#8203;NOTE: As thrombin adsorbs to glass, do not use glass flasks to prepare the stock solution or store the aliquots.</li>
	</ol>
	</li>
	<li>Preparation of thrombin-CaCl2 solution
	<ol>
		<li>Pipette 100 &#181;L of thrombin stock solution and transfer to a 50 mL conical tube containing 8.9 mL of sterile deionized water to obtain a final concentration of 1 U/mL thrombin.</li>
		<li>Prepare a 10% (w/v) CaCl2 solution in deionized water and sterilize using a 0.2 &#181;m filter.</li>
		<li>Transfer 1.1 mL of the 10% CaCl2 solution to the conical tube containing thrombin in order to obtain a final ratio of 1:9 (CaCl2 to thrombin). 
		&#8203;NOTE: Prepare the crosslinker solution at the volume for the experiment, avoiding storage.</li>
	</ol>
	</li>
</ol>
4. Bioprinting astrocytes-laden bioink using an extrusion-based bioprinter
<ol>
	<li>Design of the neural tissue&#8203;
	<ol>
		<li>Using the G-code: construct a grid of 6 x 6 mm (squared shape) with 1 mm of distance between each bioprinted line on the X and Y-axis, and 6 layers on the Z-axis (0.2 mm between each line); set the extrusion (E) to 0.01 mm, increasing 0.001 mm at each new layer of the Z-axis; and set the printing speed (F) to 400 mm/min (Supplemental Information).</li>
	</ol>
	</li>
	<li>Bioprinter set up
	<ol>
		<li>Expose the machine to UV light for 15 min, and then wipe it down with ethanol 70%.</li>
		<li>Turn on the bioprinter using the power switch. Connect the machine to the computer through a USB cable. Open the controlling software to connect it to the bioprinter and load the file design.</li>
	</ol>
	</li>
	<li>Preparation of the bioprinting syringe
	<ol>
		<li>Transfer the astrocytes-laden gelatin/GelMA/fibrinogen bioink to a 5 mL plastic syringe using a 1,000 &#181;L pipette. 
		NOTE: Transfer slowly to avoid bubble formation.</li>
		<li>Connect a sterile 22 G blunt needle to the syringe. 
		NOTE: Leave the syringe at 4 &#176;C for 2 min.</li>
		<li>Connect the syringe to the bioprinter printhead and manually flush the bioink to remove the remaining bubbles.</li>
	</ol>
	</li>
	<li>Bioprinting 
	NOTE: The bioprinting was performed outside the laminar hood.
	<ol>
		<li>Place a 35 mm culture dish on the bioprinter table and position the needle 0.1 mm away from the culture dish surface to allow needle movement. 
		&#8203;NOTE: Use one 35 mm culture dish for each bioprinting.</li>
		<li>Press the Print button.</li>
		<li>Once the bioprinting is over, ensure that the syringe moves away from the dish. Then, close the culture dish and prepare for the cross-linking process. 
		&#8203;NOTE: The bioprinting of one construct takes approximately 1 min and 10 s.</li>
	</ol>
	</li>
	<li>Crosslinking the bioprinted construct and culture
	<ol>
		<li>Place the culture dish under UV light at 2 mW/cm2 for 2 x 60 s (up and down) for GelMA crosslinking.</li>
		<li>Under the laminar flow, transfer the bioprinted construct to a 24-well plate using a sterile spatula.</li>
		<li>Add 500 &#181;L of thrombin/CaCl2 solution and leave for 30 min to allow fibrin crosslinking.</li>
		<li>Remove the crosslinking solution and wash the construct with 2 mL of PBS 1x. Then, replace the PBS with 1 mL of astrocyte culture medium and incubate at 37 &#176;C and 5% CO2. Change the medium every 3 days.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Bioprinter</td>
			<td>3D Biotechnology Solutions</td>
			<td></td>
			<td>Extrusion-based bioprinter</td>
		</tr>
		<tr>
			<td>Blunt-tip forceps</td>
			<td>Integra Miltex</td>
			<td>6--30</td>
			<td>Forceps for brain dissection previously sterilized</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td>Protease free, fatty acid free, essentially globulin free</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>Fisher Scientific</td>
			<td>156340</td>
			<td>Culture flask T25</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Abcam</td>
			<td>ab224589</td>
			<td>DAPI staining solution</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>12500062</td>
			<td>DMEM/F-12 50/50, 1X (Dulbecco&#39;s Mod. Of Eagle&#39;s Medium/Ham&#39;s F12 50/50 Mix) with L-glutamine</td>
		</tr>
		<tr>
			<td>Dyalisis tubing</td>
			<td>Sigma-Aldrich</td>
			<td>D9527</td>
			<td>Molecular weight cut-off = 14 kDa</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>64-15-5</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>12657011</td>
			<td>Research Grade</td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma-Aldrich</td>
			<td>9001-32-5</td>
			<td>Fibrinogen crystalline powder from bovine plasma</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>9000-70-8</td>
			<td>Gelatin powder from porcine skin</td>
		</tr>
		<tr>
			<td>Hanks Buffered Salt Solution (HBSS)</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>14175095</td>
			<td>No calcium, no magnesium, no phenol red</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>114956-81-9</td>
			<td>Laminin 1-2 mg/mL L in 50 mM Tris-HCl</td>
		</tr>
		<tr>
			<td>Methacrylic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>760-93-0</td>
			<td>For GelMA preparation</td>
		</tr>
		<tr>
			<td>Needle 22G</td>
			<td>Fisher Scientific</td>
			<td>NC1362045</td>
			<td>Sterile blunt needle</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Corning Incorporated</td>
			<td>430591, 430588</td>
			<td>Sterile petri dishes of 35 and 100 mm</td>
		</tr>
		<tr>
			<td>Photoinitiator</td>
			<td>Sigma-Aldrich</td>
			<td>106797-53-9</td>
			<td>2-Hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (PBS)</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>10010023</td>
			<td>PBS 1 x, culture grade, no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Poly-L-lysine hydrobromide mol wt 30,000-70,000</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>BD</td>
			<td>1222C84</td>
			<td>Sterile syringe</td>
		</tr>
		<tr>
			<td>Syringe filter 2 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>09-740-105</td>
			<td>Polypropylene filter for sterilization</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>9002--04-4</td>
			<td>Thrombin cristalline powder from bovine plasma</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Laboratory grade</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>15400054</td>
			<td>Trypsin no phenol red 1 x diluted in PBS</td>
		</tr>
		<tr>
			<td>Well plate</td>
			<td>Thermo Scientific</td>
			<td>144530</td>
			<td>Sterile 24-well plate</td>
		</tr>
	</tbody>
</table>

a method for 3d bioprinting murine cortical astrocytes

Source: de Melo, B. A. G., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62691">3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue.</a> J. Vis. Exp. (2021)
The video demonstrates the process of creating a 3D bioprinted construct using astrocytes suspended in a bioink solution. The bioprinted construct, after crosslinking, facilitates astrocyte adhesion and their spreading, forming neural-like tissue.

A Method for 3D Bioprinting Murine Cortical Astrocytes

1. Synthesis of gelatin methacryloyl (GelMA)

	Weigh 10 g of gelatin obtained from porcine skin and dissolve in 100 mL of PBS by letting the solution stir ...

Take an astrocyte suspension.
Add a bioink solution containing gelatin, photocrosslinkable gelatin derivatives, photoinitiators, fibrinogen, and laminin.
Transfer the suspension to a syringe with a blunt needle.
Chill the syringe for bioink gelling. Connect the syringe to the bioprinter&#39;s printhead.
Position the needle appropriately to the culture plate. Initiate bioprinting with set parameters and the design.
Astrocyte-containing bioink is pushed out of the needle, and is deposited in layers onto the plate, forming a three-dimensional structure with trapped astrocytes.
Expose the bioprinted construct to ultraviolet light. The photoinitiator aids in gelatin derivative crosslinking, enhancing bioconstruct stability.
Treat with thrombin and calcium ions.
Thrombin, with calcium ions, converts fibrinogen into fibrin, assembling into a stable 3D biopolymer network.
Discard the solution and wash with buffer to remove the crosslinking agents.
Add astrocyte media and incubate. Laminin aids astrocyte adhesion and spreading, forming a 3D neural-like tissue rich in astrocytes.

a-method-for-3d-bioprinting-murine-cortical-astrocytes

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

55 Views. Fonte: de Melo, B. A. G., et al., Bioprinting 3D di astrociti corticali murini per l'ingegnerizzazione di tessuti simili a neurali. J. Vis. Exp. (2021) Il video mostra il processo di creazione di un costrutto biostampato in 3D utilizzando astrociti sospesi in una soluzione bioink. Il costrutto biostampato, dopo la reticolazione, facilita l'adesione degli astrociti e la loro diffusione, formando un tessuto simile a quello neurale. 

Video: Un metodo per la biostampa 3D di astrociti corticali murini - Concept

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

A base di seta-collagene Engineered Modello 3D del tessuto neurale polarizzata

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Ricerca

JoVE Journal

Bioingegneria

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

Microcircuito sinaptica modellazione con 3D Cocultures di astrociti e neuroni da cellule staminali pluripotenti umane

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Biologia dello sviluppo

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

Bioinchiostro a matrice extracellulare derivato dal tessuto pancreatico per la stampa di costrutti di tessuto pancreatico 3D Cell-Laden

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Method for 3D Bioprinting Murine Cortical Astrocytes

Trascrizione

A Method for 3D Bioprinting Murine Cortical Astrocytes