Name: engineered 3d silk-collagen-based model of polarized neural tissue
Uploaded: 2015-10-23T14:00:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).

The brain tissue isolation protocol was approved by the Tufts University Institutional Animal Care and Use Committee and complies with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institutional Animal Care and Use Committee B2011-45).
1. Silk Scaffold Preparation
<ol>
	<li>Preparation of silk solution from Bombyx mori cocoons as described previously19,20.
	<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 bring it to 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, also 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>In order 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 immerse all the fibroin fibers using spatula. 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. Pour the supernatant into a fresh tube after each centrifugation step 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. 
		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 granules sized 500-600 &#181;m, which will be used in later steps. Discard the granules sized below 500 &#181;m and above 600 &#181;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 content 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>When ready, cut out the scaffolds with 5 mm diameter biopsy punch. 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 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 as previously described by Pacifici and Peruzzi27after 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 equal volume of 1 mg/ml soybean protein.</li>
	<li>Triturate the cortices using 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>Re-suspend 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. Using sterile forceps place the scaffolds in a 96-well cell culture plate allocating one scaffold per well. (10 min)</li>
		<li>Immerse the scaffolds in cell culture medium to equilibrate them prior to 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 for 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 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 of medium.</li>
		<li>Using sterile forceps transfer the scaffolds to the empty wells on the well plate 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 polymerization of collagen.</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>
4. Microscopy Analysis
<ol>
	<li>Live/dead staining (1 hr)
	<ol>
		<li>Prepare stock solution of propidium iodide (PI) 1 mg/ml (1.5 mM) in distilled water.</li>
		<li>Prepare stock solution of fluorescein diacetate (FDA) 2 mg/ml in acetone.</li>
		<li>Prepare working solution containing 10 &#181;M PI and 0.15 &#181;M FDA diluted in PBS. Pre-warm the solution to 37 &#176;C in a water bath.</li>
		<li>Wash the cells with pre-warmed PBS to remove the serum esterases. Aspirate the PBS.</li>
		<li>Apply the pre-warmed working solution on the cells for 2 min and wash it with PBS.</li>
		<li>Use epifluorescent microscope to image the cells (PI ex &#955;=490 nm, em &#955;=570 nm; FDA ex &#955;=490 nm, em &#955;=514 nm). The stain remains stable over 40 min. Prolonged staining may result in unspecific staining resulting from PI uptake by the cells and co-localization of PI/FDA in cells.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>At the desired time point of culture fix the cells with 4% paraformaldehyde (PFA) for 30 min at RT. Due to the toxicity of the PFA fumes the fixing step should be performed in the chemical fume hood.</li>
		<li>Wash scaffolds with 3x PBS. Stopping point: The PFA-fixed constructs can be stored in PBS at 4 &#176;C for several days before proceeding with further steps.</li>
		<li>Incubate the scaffolds in 0.2% Triton, 0.25% bovine serum albumin (BSA) in PBS containing primary antibody O/N at 4 &#176;C.</li>
		<li>The following day discard the staining solution and wash the scaffolds 3 x 30 min with PBS to remove the unbound primary antibody.</li>
		<li>Incubate the samples with secondary antibody diluted in 0.2% Triton, 0.25% BSA in PBS for 2 hr at RT.</li>
		<li>Remove the staining solution and wash the scaffolds 3 x 30 min with PBS to remove the unbound secondary antibody.</li>
		<li>Image the scaffolds using a confocal microscope with 20X objective by taking 100 &#181;m z-sections of the sample with 1 &#181;m step. To visualize the thin neurites the image resolution should be of minimum 1,024 x 1,024 pixels.</li>
	</ol>
	</li>
	<li>RNA, DNA and protein isolation 
	Note: RNA, DNA and protein isolation is performed using a commercial AllPrep DNA/RNA/Protein Mini Kit according to the manufacturer instructions.
	<ol>
		<li>Using sterile forceps transfer the scaffolds out of culture plate to a 2 ml sterile tube. Place one scaffold per tube.</li>
		<li>Add 200 &#181;l of RLT buffer to each tube.</li>
		<li>Disrupt the construct by fragmenting it with sterile microscissors.</li>
		<li>To homogenize the disrupted tissue, transfer the content of the tube to the spin column. Spin for 2 min at full speed in a microcentrifuge. The lysate is homogenized as it passes through the spin column.</li>
		<li>Collect the flow through and use it immediately to isolate RNA, DNA and protein following the manufacturer protocol.</li>
		<li>Quantify the yield of the nucleic acids any other compatible method (e.g., NanoDrop).</li>
		<li>Quantify the protein yield using BCA Protein Assay according to the manufacturer instructions. Measure the outcome of assay using microplate reader at wavelength 562 nm. 
		Note: The expected yield of nucleic acids and protein per construct in relation to the cell density is shown in Figure 4.</li>
	</ol>
	</li>
</ol>

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Publication of this video-article is sponsored by Corning.

Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.

The central nervous system (CNS) can be affected by a variety of disorders involving vascular, structural, functional, infectious or degenerative. An estimated 6.8 million people die every year as a result of neurological disorders, which represents a growing socioeconomic burden worldwide1. However, only few of the disorders have available treatments. Therefore, there is a critical need for innovative therapeutic strategies for patients suffering from neurological disorders. Unfortunately, many CNS-targeted therapeutics fail during clinical trials, in part due to the utilization of inadequate pre-clinical research models, which do not allow assessment of acute and chronic impacts with physiologically-relevant functional readouts.
Despite significant research efforts over the past decades, there is a vast amount unknown about the structure and function of CNS. In order to gain more knowledge, animal models are frequently used to model pathological states, such as traumatic brain injury (TBI) or dementia, especially in pre-clinical studies. However, animals differ significantly from humans both in anatomy of CNS, as well as in function, gene expression and metabolism2-4. On the other hand, 2D in vitro cultures are the common method to investigate cell biology and are routinely used for drug discovery. However, 2D cell cultures lack the complexity and physiological relevance as compared to human brain5-7. While there is no substitute for the low cost and simplicity of 2D cell culture studies or the complexity provided by animal models, 3D tissue engineering could generate improved research models to close the gap that exists between the 2D in vitro and in vivo approaches. 3D tissue engineering provides more physiologically-relevant experimental conditions achieved by 3D cell-cell interactions and extracellular cues provided by the biomaterial scaffolds. Despite the significant evidence behind the value of 3D cultures, there are currently only a few 3D CNS tissue models such as stem cell-derived organoid cultures8-10, neurospheroids11 and dispersed hydrogel cultures12,13. Advanced technical methods including multilayer lithography14, and 3D printing15 have been utilized for studying lung, liver, and kidney tissue. However, there is lack of 3D CNS models that allow for compartmentalized neuronal growth, such as mimicking the cortical architecture and biology. Separated growth of neurites from neuronal cell bodies has been previously demonstrated in 2D cultures by using microfabrication16,17 allowing the study of axon tract tracing, calcium influx, network architecture and activities. This idea inspired us to develop a 3D polarized neural tissue where cell bodies and axonal tracts are located in different compartments mimicking the layered architecture of the brain18. Our approach is based on the use of unique silk scaffold design which accommodates high density of cells in a confined volume and allows outgrowth of dense axonal network into a collagen gel. Here we demonstrate the full assembly procedure of brain-like tissue including the scaffold fabrication and neuronal culture.

<table><tbody><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</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&amp;nbsp;</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>Centrifuge 5804 R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Sieve</td><td>Fisher Scientific</td><td></td><td>No. 270, No. 35, No. 30</td></tr><tr><td>PTFE mold</td><td></td><td></td><td>made in house (&lt;strong&gt;Figure 1&lt;/strong&gt;) 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&amp;#160;mm</td><td>World Precision Instrument</td><td>501909</td><td></td></tr><tr><td>Biopsy Punch 2&amp;#160;mm</td><td>World Precision Instrument</td><td>501908</td><td></td></tr><tr><td>poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407-5MG</td><td>final concentration 10&amp;#160;&amp;#956;g/ml, dissolved in water</td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D1283-500ML</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>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&amp;#160;mg/ml</td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>warm up to 37&amp;#160;&amp;deg;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&amp;#160;mg</td><td>Corning</td><td>354236</td><td>final concentration 3&amp;#160;mg/ml</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S2770</td><td>corrosive</td></tr><tr><td>Propidium Iodide</td><td>Sigma-Aldrich</td><td>P4170-10MG</td><td>toxic</td></tr><tr><td>Fluorescein Diacetate</td><td>Sigma-Aldrich</td><td>F7378-5G</td><td></td></tr><tr><td>Olympus MVX10</td><td>Olympus</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>toxic, final concentration 4%</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906-10G</td><td></td></tr><tr><td>anti-&amp;beta;IIITubulin antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T2200</td><td>rabbit 1:500</td></tr><tr><td>anti-rabbit Alexa-546</td><td>Molecular Probes</td><td>A11010</td><td>goat 1:250</td></tr><tr><td>goat serum</td><td>Sigma-Aldrich</td><td>G9023-5ML</td><td></td></tr><tr><td>Leica SP2 confocal microscope</td><td>Leica</td><td></td><td>objective 20X</td></tr><tr><td>QIAshredder</td><td>Qiagen</td><td>79654</td><td></td></tr><tr><td>AllPrep DNA/RNA/Protein Mini Kit</td><td>Qiagen</td><td>80004</td><td></td></tr><tr><td>Ethyl alcohol, Pure</td><td>Sigma-Aldrich</td><td>E7023&amp;nbsp;</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>63689</td><td></td></tr><tr><td>NanoDrop 2000c/2000 UV-Vis Spectrophotometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>BCA protein assay</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Plate reader Spectramax M2</td><td>Molecular Devices</td><td></td><td>absorbance 562 nm</td></tr><tr><td>Micro Scissors, Economy, Vannas-type</td><td>TedPella</td><td>1346</td><td></td></tr></tbody></table>

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.

Solid silk sponges precut to the donut shape represent a unique and simple idea to achieve a compartmentalized architecture resembling neural tissue (Figure 2A). The highly porous structure of the scaffold with the pore size of about 500 &#181;m results in exceptionally high surface area allowing the seeding and growth of high cell density within a small volume (2x107/ml) (Figure 2B). Additionally, the high porosity of the scaffold allows for unrestricted diffusion of nutrients and wastes resulting in superior viability of dense cell cultures over extended time frames. Upon seeding the neurons rapidly and uniformly attach to the surface of the scaffold pores.
After cell attachment is complete and cells are equilibrated on the silk substrate, the sponge scaffold is filled with the soft collagen matrix in order to provide a 3D environment for axonal network formation (Figure 3). In the absence of the collagen matrix the compartmentalization of axons and cell bodies is not possible as neurons grow extensions only on the surface of the pores resulting in mixed networks as found with 2D surfaces (Figure 3B). In contrast, the collagen gel provides a meshwork which supports extensive axonal outgrowth in 3D, while neuronal cell bodies remain attached to the silk scaffold (Figure 3C). This growth pattern leads to compartmentalization of cell bodies and pure axonal networks (Figure 3D), which resembles the gray and white matter of the cortex in the brain.
Apart from microscopy, the constructs can be evaluated with a variety of other methods18, depending on the question behind the experiment. For example, the isolation of DNA, RNA and protein from the constructs can be easily performed using commercial kits (Figure 4). The yield of nucleic acids and protein per construct depends on the number of cells initially seeded on the silk scaffolds. The more cells seeded the higher the yield of DNA, RNA and protein.
<img alt="Figure 1" src="/files/ftp_upload/52970/52970fig1.jpg" /> 
Figure 1. PTFE mold used for silk sponge scaffold preparation. The dimensions: 10 cm diameter, 2 cm height. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52970/52970fig2.jpg" /> 
Figure 2. 3D brain-like tissue model.&#160;(A) Porous silk sponge scaffold. (B) Live/dead staining of neurons at day 1 upon seeding on silk scaffold before collagen embedding (green-live cells, red-dead cells). Panels on right side show the magnification of area captured in red frames. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52970/52970fig3.jpg" /> 
Figure 3. Neuronal compartmentalized outgrowth in the 3D brain-like tissue model.&#160;(A) Scaffold compartments: Red-cell body compartment, Blue-axonal compartment. (B) Neuronal growth pattern at day 1 upon seeding before collagen embedding. (C). Neuronal outgrowth at day 7 upon seeding in the cell body compartment. (D) Neuronal outgrowth at day 7 upon seeding in the axonal compartment. Green &#8212; &#946;IIITubulin. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52970/52970fig4.jpg" /> 
Figure 4. Expected yield of (A) RNA and DNA, and (B)&#160;protein per construct in relation to the amount of cells seeded per scaffold (&#177;SD, n=5). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

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

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.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

12.4K Views.  Tufts University. 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.

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

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Cancer Research

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Neuroscience

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

3D Bioprinted Cocultures of iPSC-Derived Neurons and Astrocytes

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models&#160;do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

Author Spotlight: Advancing 3D Cell Modeling &#8211; A High-Throughput Approach for Neural Cocultures 

Here, we present a protocol to produce 3D-bioprinted cocultures of iPSC-derived neurons and astrocytes. This coculture model, generated within a hydrogel scaffold in 96- or 384-well formats, demonstrates high post-print viability and neurite outgrowth within 7 days and shows the expression of maturity markers for both cell types.

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development ...

Applications of 3D-Printing for Micro-Scale Neuronal Cell Culture Devices

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Author Spotlight: Modular Neuronal Networks for Analyzing Brain Functions

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

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

Source: Chwalek, K. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

Opracowanie zmodyfikowanego modelu 3D spolaryzowanej tkanki nerwowej na bazie jedwabiu

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

	...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

Source: Sharif, R., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56308">Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea.</a> J. Vis. Exp. (2018)
This video demonstrates a technique to generate a three-dimensional co-culture model system of primary human corneal fibroblasts and differentiated neuronal cells. This co-culture system can be used to study the stromal-nerve interactions.

Generowanie modelu 3D kokultury ludzkich fibroblastów i neuronów rogówki

1. Primary HCFs (human corneal fibroblasts) Culture and 3D Constructs Assembly

	To assemble 3D constructs from explants previously grown in EMEM 10% FBS ...

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

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Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Opracowanie zmodyfikowanego modelu 3D spolaryzowanej tkanki nerwowej na bazie jedwabiu

Generowanie modelu 3D kokultury ludzkich fibroblastów i neuronów rogówki