developing an engineered silk-collagen-based 3d model of polarized neural tissue

Developing an Engineered Silk-Collagen-Based 3D Model of Polarized Neural Tissue

Inside a cell culture hood, place a pair of sterilized forceps, cell culture media, and the autoclaved scaffolds. Remove a 96-well plate from its wrapping and place inside. To seed the scaffolds, first, place one sterile scaffold per well in the 96-well plate. Add cell culture medium to immerse the scaffolds, and incubate at 37 degrees Celsius in a tissue culture incubator to equilibrate them for at least 30 minutes.
Following incubation, aspirate the excess medium from the scaffolds. Then, add 2 times 10 to the 6 rat cortical neuronal cells in 100 microliters of neurobasal medium per scaffold. Return the plate to the incubator, and leave overnight to allow the cells to attach to the scaffold. On the following morning, carefully aspirate the non-attached cells, and replace with 200 microliters of fresh cell culture medium. Incubate at 37 degrees Celsius for a brief period.
Next, aspirate the medium from the wells. Using sterile forceps, transfer the scaffolds containing cells to empty wells of the 96-well plate. Then to each scaffold, add 100 microliters of freshly diluted ice-cold 3 milligrams per milliliter rat tail collagen solution.
Return the cells to the 37 degrees Celsius incubator to allow polymerization of collagen for 30 minutes. Remove the plate from the incubator, and add 100 microliters of pre-warmed cell culture medium per well. Return the plate to the incubator for the desired period of cell growth, replacing half of the medium from each well with fresh medium every day for up to one week.

developing-an-engineered-silk-collagen-based-3d-model-polarized

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Silk Scaffold Preparation
<ol>
	<li>Preparation of silk solution from Bombyx mori cocoons
	<ol>
		<li>Cut each cocoon into 8 equal pieces using scissors. Use about 11 cocoons for 5 g of fragmented cocoons. (15 min)</li>
		<li>Prepare 2 L of 0.02 M Na2CO3 solution and boil using a hot plate. (15 min)</li>
		<li>Weigh 5 g of fragmented cocoons and boil them in Na2CO3 solution for 30 min. Stir the boiling silk every couple of minutes with a spatula. This step, called de-gumming, purifies silk fibroin from hydrophilic proteins, sericins. (30 min)</li>
		<li>Wring the fibroin out by hand and rinse it in distilled water at least three times to wash out any remaining sericin and chemicals. (5 min)</li>
		<li>Place the wet fibroin on a petri dish and dry the fibroin extract in the flow hood O/N.</li>
		<li>The next day, weigh the dry fibroin mass and place the fibroin in a glass beaker. (15 min)</li>
		<li>To dissolve the fibroin in 9.3 M LiBr solution, calculate the required volume (in ml) of LiBr by multiplying the mass of dry fibroin by 4. Slowly pour LiBr solution over the silk fibroin and use a spatula to immerse all the fibroin fibers. Cover the beaker to prevent evaporation and place it at 60 &#176;C for 4 hr to allow the fibers to dissolve. (15 min)</li>
		<li>Using the syringe, collect the fibroin solution from the beaker and load it into the MWCO 3,500 dialysis cassettes. Perform dialysis against distilled water for 48 hr. Change the water every couple of hours.</li>
		<li>Using the syringe, collect the fibroin solution from the cassettes into 50 ml conical tubes and centrifuge twice at 9,000 rpm (~12,700 x g) at 4 &#176;C for 20 min. After each centrifugation step, pour the supernatant into a fresh tube and discard the pellet. (40 min)</li>
		<li>Measure the concentration of the fibroin by estimating the dry weight. Pour 1 ml of silk solution into a weigh boat. Dry the sample in the oven at 60 &#176;C for 2 hr. Weigh the dry silk fibroin and calculate the concentration of silk fibroin solution by multiplying the obtained weight by 100. The expected concentration of silk solution is 6-9% (w/v).</li>
		<li>Adjust the silk concentration to 6% (w/v) by diluting it in distilled water. 
		&#8203;Stopping point: The liquid silk fibroin can be stored at 4 &#176;C for up to one month in a closed container.</li>
	</ol>
	</li>
	<li>Preparation of porous scaffolds from silk solution.
	<ol>
		<li>Sieve the granular NaCl to separate the 500-600 &#956;m granules, which will be used in later steps. Discard the granules sized below 500 &#956;m and above 600 &#956;m. (15 min)</li>
		<li>Pour 30 ml of 6% silk solution into the polytetrafluoroethylene (PTFE) mold (Figure 1). Gently scatter 60 g of sieved NaCl over the silk. Tap the container to obtain a uniform layer of salt. Incubate 48 hr at RT to polymerize the silk.</li>
		<li>Place the scaffold-containing PTFE mold in the oven at 60 &#176;C for 1 hr to finalize the polymerization and evaporate any remaining liquid.</li>
		<li>Place the contents of the PTFE mold in a beaker containing 2 L of distilled water for 48 hr to leach out the salt. Change the water 2-3 times per day. Remove the sponge scaffolds from the molds when the salt is completely leached out. 
		Stopping point: The sponges can be stored immersed in water at 4 &#176;C in a closed container to prevent the scaffolds from dehydration.</li>
		<li>Cut out the scaffolds with a 5 mm diameter biopsy punch when ready. Pre-cut the scaffolds to reach about 2 mm in height. Punch out the center of the scaffold with the 2 mm diameter biopsy punch (Figure 2A). (1 hr)</li>
		<li>Autoclave the scaffolds immersed in water to sterilize them (wet cycle, 121 &#176;C, 20 min). 
		Stopping point: The sponges can be stored immersed in water at 4 &#176;C in a closed container to prevent the scaffolds from dehydration.</li>
		<li>Before the planned cell seeding, immerse the scaffolds in a sterile 0.1 mg/ml poly-D-lysine (PDL) solution. Incubate for 1 hr at 37 &#176;C.</li>
		<li>Wash the scaffolds 3x with phosphate-buffered saline (PBS) to remove non-bound PDL. (30 min)</li>
	</ol>
	</li>
</ol>
2. Isolation of Rat Cortical Neurons
<ol>
	<li>Dissect cortices from embryonic day 18 (E18) Sprague-Dawley rats after the approved animal protocol is obtained. (2 hr)</li>
	<li>Incubate 10 cortices in 5 ml of 0.25% trypsin with 0.3% DNase I (from bovine pancreas) for 20 min at 37 &#176;C.</li>
	<li>Inactivate the trypsin by adding 1 mg/ml of soybean protein.</li>
	<li>Triturate the cortices using a 10 ml Pasteur pipette by pipetting up and down 20 times until a single cell suspension is generated. Be gentle and avoid air bubble formation. (10 min)</li>
	<li>Centrifuge the cell suspension at 127 x g for 5 min.</li>
	<li>Resuspend the cell pellet in 10 ml of culture medium (Neurobasal medium, 1x B27 supplement, 1x Glutamax, 1% penicillin/streptomycin). Count the cells. The expected cell concentration is about 2x107/ml. (20 min)</li>
</ol>
3. Construct Assembly and Culture
<ol>
	<li>Scaffold seeding with cells
	<ol>
		<li>Move the sterile scaffolds and all the required utensils inside a cell culture hood. Place the scaffolds in a 96-well cell culture plate using sterile forceps, allocating one scaffold per well. (10 min)</li>
		<li>Immerse the scaffolds in the cell culture medium to equilibrate them before cell seeding. Incubate for 1 hr at 37 &#176;C. (10 min)</li>
		<li>Aspirate the excess of the medium from the scaffolds.</li>
		<li>Apply 100 &#181;l of cell suspension/scaffold. (10 min)</li>
		<li>Incubate the cells at 37 &#176;C O/N to allow cell attachment to the scaffold.</li>
		<li>The following morning, aspirate the non-attached cells and apply 200 &#181;l/well of fresh culture medium. (10 min)</li>
	</ol>
	</li>
	<li>Scaffold embedding with collagen matrix. (2 hr)
	<ol>
		<li>Place 10x PBS, water, 1 N NaOH, and rat tail I collagen on ice. Prepare a working solution of collagen as per the manufacturer&#39;s instructions. Maintain on ice until the cell-seeded constructs are ready (up to 1 hr).</li>
		<li>Remove the cell-seeded silk constructs from the incubator and aspirate the excess medium.</li>
		<li>Transfer the scaffolds to the empty wells on the well plate using sterile forceps and immerse each scaffold in 100 &#181;l of 3 mg/ml collagen solution. Place the tissue culture plate back in the incubator for 30 min to allow collagen polymerization.</li>
		<li>Apply 100 &#181;l of pre-warmed cell culture medium/well. Culture the constructs for one week, changing the medium every day by replacing only half the volume of the medium.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Centrifuge 5804 R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>final concentration 10 &#956;g/ml, dissolved in water</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>warm up to 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>B27 supplement 50x</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>final concentration 3 mg/ml</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>corrosive</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1283-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2CO3</td>
			<td>Sigma-Aldrich</td>
			<td>223530</td>
			<td></td>
		</tr>
		<tr>
			<td>LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td></td>
		</tr>
		<tr>
			<td>MWCO 3500 dialysis cassettes</td>
			<td>Thermo Fisher</td>
			<td>66110</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td></td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>Fisher Scientific</td>
			<td>No. 270, No. 35, No. 30</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE</td>
			<td></td>
			<td></td>
			<td>mold made in house (Figure 1) 10 cm diameter, 2 cm height</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71382</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch 5 mm</td>
			<td>World Precision Instrument</td>
			<td>501909</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch 2 mm</td>
			<td>World Precision Instrument</td>
			<td>501908</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;T4049-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td>final concentration 0.3%</td>
		</tr>
		<tr>
			<td>Soybean protein</td>
			<td>Sigma-Aldrich</td>
			<td>T6522-100MG</td>
			<td>final concentration 1 mg/ml</td>
		</tr>
	</tbody>
</table>

Source: Chwalek, K. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/52970">Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue.</a>&#160;J. Vis. Exp. (2015).
This video showcases the creation of a 3D polarized neural tissue model utilizing silk-collagen scaffolds. It details the process of seeding neuronal cells on porous silk scaffolds, incubating for cell attachment, embedding the scaffold with collagen, and underscores the significance of the supportive collagen matrix in forming a 3D polarized neural tissue model.

<img alt="Figure 1" src="/files/ftp_upload/22396/22396_Figure1.jpg" /> 
Figure 1. PTFE mold used for silk sponge scaffold preparation. The dimensions: 10 cm diameter, 2 cm height.
<img alt="Figure 2" src="/files/ftp_upload/22396/22396_Figure2.jpg" /> 
Figure 2. 3D brain-like tissue model. (A) Porous silk sponge scaffold. (B) Live/dead staining of neurons at day 1 upon ...

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Silk Scaffold Preparation

	...

Place donut-shaped, poly-D-lysine-coated, porous silk scaffolds into a multi-well plate.&#160;
Add culture media to hydrate the scaffolds, facilitating cell attachment.
Aspirate excess media. Seed the scaffolds with a neuronal cell suspension and incubate.
The coated surface and the scaffold pores provide a large surface area for high-density cell attachment and facilitate efficient nutrient and waste diffusion.
Remove the non-attached cells and add collagen, an extracellular matrix protein.
Incubate for collagen polymerization. This process embeds the cells within a gel-like 3D matrix on the porous scaffold.
Add media. Culture, and regularly replace half of the culture media to replenish nutrients without disturbing the cells.
Within the culture, the neuronal cell bodies remain anchored to the silk scaffold while the axons grow through the collagen matrix, forming 3D axonal networks.
This compartmentalized cell body and axonal localization results in the formation of a 3D brain-like polarized neural tissue model.

22 조회수. 편광 신경 조직의 엔지니어링된 실크-콜라겐 기반 3D 모델 개발

비디오: 편광 신경 조직의 엔지니어링된 실크-콜라겐 기반 3D 모델 개발 - 실험

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

연골과 피부 조직 아날로그 소설 수동 Bioink Precellularization에 대 한 단위 기술 혼합을 이용 하 여 Bioprinting

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

연구

JoVE Journal

생체공학

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

신경과 같은 조직을 위한 Murine 피질 성상 세포의 3D 생물 인쇄

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

공학적 심장 조직의 데이터 수집 및 모델 처리량을 개선하기 위한 바이오리액터 설계

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

Developing an Engineered Silk-Collagen-Based 3D Model of Polarized Neural Tissue

내레이션 대본

Developing an Engineered Silk-Collagen-Based 3D Model of Polarized Neural Tissue