Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number R15AR064481, the National Science Foundation (CMMI-1404716), as well as the Arkansas Biosciences Institute.

1. Production of Extracellular Matrix Using Sacrificial Hollow Fiber Membranes
CAUTION: N-methyl-2-pyrrolidone is an irritating solvent and reproductive toxicant. Exposure to NMP may cause irritation to the skin, eyes, nose, and throat. Solvent-resistant personal protective equipment should be used when handling NMP. Use of NMP should be performed within a fume hood.
<ol>
	<li>Preparation of polysulfone polymer solution for hollow fiber membranes
	<ol>
		<li>Weigh 70 g of polysulfone pellets (molecular weight of 35 kD) in a weigh boat using an analytical balance.</li>
		<li>Transfer 314 mL (323.3 g) of N-methyl-2-pyrrolidone (NMP) to a clean borosilicate flask.</li>
		<li>Place a stir bar in the flask with NMP and place flask onto stirrer. Set the stirrer to a moderate rate of rotation.</li>
		<li>Use a dry funnel to slowly insert the prepared 70 g of polysulfone into the flask with NMP.</li>
		<li>Allow the polymer &#8220;dope&#8221; solution to stir for three days at room temperature, or until homogenized.</li>
	</ol>
	</li>
	<li>Setting concentricity and cleaning of the hollow fiber membrane spinneret 
	NOTE: Spinnerets are commercially available; correct spinneret dimensions are critical to successful membrane fabrication and are listed in the Table of Materials. Handle the spinneret very gently, as the spinneret needle is particularly fragile.
	<ol>
		<li>Use a screwdriver or drill with an appropriate screwdriver bit to carefully disassemble the spinneret.</li>
		<li>Gently flow acetone through the inlet and outlet of the spinneret, collecting any acetone and residual polymer in a glass beaker for disposal.</li>
		<li>Secure the upper body of the spinneret with the needle facing upward in a table vise.</li>
		<li>Place the outlet (bottom) body of the spinneret onto the upper body.</li>
		<li>Move the outlet body of the spinneret laterally while holding the upper body fixed on the vise until the spinneret needle is directly in the center of the outlet body.</li>
		<li>Carefully and slowly reinsert the screws connecting the two bodies of the spinneret while ensuring that the needle visible at the spinneret outlet remains centered.</li>
	</ol>
	</li>
	<li>Fabrication of polysulfone hollow fiber membranes
	<ol>
		<li>Prepare a &#8220;bore solution&#8221; containing 45 mL of N-methyl-2-pyrrolidone and 255 mL of deionized water, forming a 15% mixture of NMP with deionized water.</li>
		<li>Mount a concentric hollow fiber membrane spinneret 8 cm above the surface of a room-temperature tap water bath.</li>
		<li>Connect the polymer inlet of the spinneret and the bore inlet of the spinneret to two separate steel pressure vessels having an inner volume of at least 350 mL. Both vessels must have check valves at their outlets.</li>
		<li>Connect each of the pressure vessels to the regulators of separate N2 gas cylinders</li>
		<li>Use a funnel to insert the prepared dope solution (~315 mL) into the pressure vessel connected to the polymer inlet of the spinneret. Ensure that the top of the vessel is tightly sealed.</li>
		<li>Use a funnel to insert the prepared bore solution (300 mL) into the pressure vessel connected to the bore inlet of the spinneret. Ensure that the top of the vessel is tightly sealed.</li>
		<li>Open the check valve for the bore vessel first, then the polymer vessel second. If the spinneret is sufficiently clean, the bore solution will begin to stream out of the spinneret outlet.</li>
		<li>Pressurize both vessels to 1 PSI. Visually confirm that both polymer and bore solutions are exiting the spinneret, with a continuous white filament precipitating as the combined streams contact the water bath.</li>
		<li>Use long forceps to guide the nascent fiber under the rollers in the center of the bath, and then wind the nascent fiber around a rotating wheel connected to a motor.</li>
		<li>Set the take-up wheel and motor to rotate at a rate of approximately two meters per minute.</li>
		<li>Make minor adjustments to the regulators or take-up wheel speed as needed until a steady-state flow is achieved. The nascent fiber should form a 90&#176; angle with the surface of the water bath.</li>
		<li>Close the N2 cylinder regulators and pressure vessel check valves and disengage the take-up wheel motor after all polymer and bore solution has been extruded from the vessels.</li>
		<li>Remove the prepared hollow fiber membranes and place them into a deionized water bath for three days, exchanging deionized water once per day to remove residual solvent.</li>
		<li>Sterilize the hollow fiber membranes by autoclaving them at 121 &#176;C for 30 minutes or by immersing them for one day in 70% ethanol.</li>
	</ol>
	</li>
	<li>Cell seeding of hollow fiber membranes 
	NOTE: All cell culture procedures should be performed within a biosafety cabinet.
	<ol>
		<li>Treat the hollow fiber membranes with a 20 &#181;g/mL solution of bovine plasma fibronectin in PBS (pH = 7.4) to promote cell attachment.
		<ol>
			<li>Weigh 1 mg of powdered bovine plasma fibronectin in a weigh boat and dissolve it in 1 mL of sterile PBS (pH = 7.4) in a sterile 1.5 mL microcentrifuge tube.</li>
			<li>Incubate the 1 mg/mL bovine plasma fibronectin solution in the incubator for 30 minutes. Add the solution to 49 mL of sterile PBS in a sterile 50 mL conical centrifuge tube.</li>
			<li>Incubate the fibers in the prepared 50 mL of bovine plasma fibronectin in an incubator at 37 &#176;C with 5% CO2 for 1 hour. Recollect the fibronectin solution for later use and store in a sterile 50 mL conical centrifuge tube at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Use sterile microscissors to cut the fibers to desired length (&#60;6 cm).</li>
		<li>Seed fibroblast cells directly into the lumina of the hollow fiber membranes at a seeding density of 100,000 cells per fiber using a 21-gauge needle with 1 mL syringe. 
		NOTE: A practicable target for preparing the cell-suspension for loading in the syringe is a suspension density of 100,000 cells for every 30 microliters of medium.</li>
		<li>Place six seeded fibers into a 6 cm diameter Petri dish. Allow the seeded fibers to incubate at 37 &#176;C with 5% CO2 for five minutes.</li>
		<li>Remove the seeded fibers from incubation and culture them for up to 3 weeks in DMEM/F-12 containing final concentrations of 50 &#181;g/mL L-ascorbic acid and 150 &#181;g/mL L-ascorbic acid 2-phosphate (freshly prepared and sterile filtered), 5 ng/mL TGF-&#946;1 (from sterile filtered aliquots stored at -20 &#176;C), and 1% penicillin-streptomycin in a 6 cm Petri dish within a 5% CO2 incubator at 37 &#176;C. Exchange cell medium every two days.</li>
	</ol>
	</li>
	<li>Extraction of extracellular matrix from cultured hollow fiber membranes
	<ol>
		<li>Place cultured fibers into individual scintillation vial using forceps.</li>
		<li>Place up to 5 mL of NMP into each vial using a glass pipette.</li>
		<li>Immerse the hollow fibers in NMP, performing a total of three exchanges of NMP. Slowly aspirate the old NMP using a 1 mL pipette to prevent tearing of extracted ECM. 
		NOTE: When adding fresh NMP, it is helpful to tilt the vial and allow the NMP run down the sides, otherwise the remaining ECM scaffold is subjected to shear which may cause tearing.</li>
		<li>Remove NMP and rinsed the resulting ECM thread 3 times in deionized water.</li>
		<li>Cut a 1/32-inch thick sheet of silicone rubber to a length of 3 inches, width of 1 inch and cut an 8 mm by 4 mm rectangular mold in the center of the length of rubber.</li>
		<li>Place the prepared piece of rubber onto a standard 3-inch by 1-inch microscopy slide and autoclave at 121 &#176;C for 30 minutes.</li>
		<li>Lay each ECM fiber side by side in the 8 mm by 4 mm silicone mold until there is no visible open space.</li>
		<li>Place the mold into a 50 mL conical centrifuge tube and freeze at -80&#176;C until completely frozen.</li>
		<li>Lyophilize frozen ECM mesh overnight or until completely dry. Store the mesh at 4&#176;C until ready for decellularization.</li>
	</ol>
	</li>
</ol>
2. Decellularization of Extracellular Matrix
<ol>
	<li>Prepare a solution of 1% (w/w%) sodium dodecyl sulfate (SDS) in deionized water.</li>
	<li>Incubate extracted extracellular matrix in the mold in 1% SDS for 24 hours at room temperature on a rocker with gentle agitation.</li>
	<li>Rinse extracted extracellular matrix three times in sterile phosphate-buffered saline (PBS) with gentle agitation, using 3 mL of PBS per rinse. 
	NOTE: Rinses should be performed gently to minimize tearing of prepared scaffolds.</li>
	<li>Prepare 1 L of DNAse/RNAse digestion buffer. 
	NOTE: DNAse/RNAse digestion buffer may be stored for several months at 4&#176;C.
	<ol>
		<li>Weigh 1.54 g of Tris HCl, 308 mg of MgCl2, and 56 mg of CaCl2 in separate weigh boats.</li>
		<li>Combine Tris HCl, MgCl2, and CaCl2 with 1 L of deionized water in a large flask with a stir bar and stir until all components are dissolved.</li>
		<li>Sterile filter the digestion buffer with a 0.22 &#181;m filter into a sterile 1 L borosilicate bottle and store at 4&#176;C.</li>
	</ol>
	</li>
	<li>Prepare 5 mL of DNAse/RNAse digestion solution. 
	NOTE: Digestion solution should be used within 24 hours of preparation.
	<ol>
		<li>Weigh 0.125 mg (50 kU) of DNAse I in a weigh boat and add to 5 mL of digestion buffer.</li>
		<li>Add 75 &#181;L of RNAse A stock solution to the digestion buffer.</li>
	</ol>
	</li>
	<li>Fill each mold with digestion solution and incubate at 4 &#176;C for 6 hours.</li>
	<li>Aspirate the digestion solution and rinse three times in sterile PBS.</li>
	<li>Aspirate the PBS and incubate scaffolds overnight in 10% penicillin-streptomycin in PBS at 4&#176;C.</li>
	<li>Aspirate the penicillin-streptomycin and rinse scaffolds three times in sterile PBS.</li>
	<li>Place the mold into a 50 mL conical centrifuge tube and freeze at -80&#176;C.</li>
	<li>Lyophilize the decellularized ECM mesh overnight or until completely dry.</li>
	<li>Transfer the lyophilized scaffolds within a biosafety hood to a sterile container at 4&#176;C pending use.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials/Tissue+Interactions:+Possible+Solutions+to+Overcome+Foreign+Body+Response.">Biomaterials/Tissue Interactions: Possible Solutions to Overcome Foreign Body Response.</a> The AAPS Journal. 12 (2), 188-196 (2010).</li><li>Simon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+failure+of+the+tissue+engineered+porcine+heart+valve+SYNERGRAFT&#174;+in+pediatric+patients.">Early failure of the tissue engineered porcine heart valve SYNERGRAFT&#174; in pediatric patients.</a> European Journal of Cardio-Thoracic Surgery. 23 (6), 1002-1006 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FDA strengthens requirements for surgical mesh for the transvaginal repair of pelvic organ prolapse to address safety risks. , (2016).</li><li>Kartus, J., Movin, T., Karlsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-site+morbidity+and+anterior+knee+problems+after+anterior+cruciate+ligament+reconstruction+using+autografts.">Donor-site morbidity and anterior knee problems after anterior cruciate ligament reconstruction using autografts.</a> Arthroscopy: The Journal of Arthroscopic &amp; Related Surgery. 17 (9), 971-980 (2001).</li><li>Lu, H., Hoshiba, T., Kawazoe, N., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+extracellular+matrix+scaffolds+for+tissue+engineering.">Autologous extracellular matrix scaffolds for tissue engineering.</a> Biomaterials. 32 (10), 2489-2499 (2011).</li><li>Wolchok, J. 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The authors declare that they have no competing financial interests.

The processes described enable the production of bulk ECM biomaterials in vitro using hollow fiber membranes cast by a dry-jet wet spinning system allowing for inexpensive bulk production of membranes as well as standard cell culture equipment. While the membranes fabricated in this protocol are intended for use in cell culture, the system described can also be adapted for the production of membranes for separation purposes, with pore size distribution and hollow fiber dimensions tunable by varying spinneret dim...

Implantable surgical scaffolds are a fixture of&#160;wound repair,with over one million synthetic polymer meshes implanted worldwide each year for abdominal wall repair alone1. However, following implantation the synthetic materials polymers traditionally used in the fabrication of these scaffolds tend to provoke a foreign body response, resulting in inflammation deleterious to the function of the implant and scarring of tissue2. Further, as the predominant synthetic mesh materials (i.e., polypropylene) are not appreciably remodeled by the body, they are generally applicable to tissues where scarring can be tolerated, limiting their clinical usefulness toward the treatment of tissues with higher-order function such as muscle. While there are many surgical mesh products which have been applied with clinical success, recent manufacturer recalls of synthetic surgical meshes and complications from interspecies tissue implants highlight the importance of maximizing implant biocompatibility, prompting the FDA to tighten regulations on surgical mesh manufacturers3,4. Implantation of scaffolds derived from patients&#39; own tissues reduces this immune response, but can result in significant donor-site morbidity5. Extracellular matrix (ECM) scaffolds produced in vitro are a possible alternative, as decellularized ECM scaffolds exhibit excellent biocompatibility, particularly in the case of autologous ECM implants6.
Because of the limited availability of patient tissue to harvest for autologous implantation and the risk of impeding function at the donor site, the ability to produce ECM scaffolds in vitro from the culture of human cell lines or, if possible, a patient&#39;s own cells is an attractive alternative. The primary challenges in the manufacture of substantial amounts of ECM in vitro is the sequestration of these difficult-to-capture molecules. In previous work, we have demonstrated that ECM can be produced by culturing ECM-secreting fibroblasts in sacrificial polymeric foams that are dissolved after the culture period to yield ECM which can be decellularized for implantation7,8,9,10. As ECM produced in foams tend to adopt the internal architecture of the foams, hollow fiber membranes (HFMs) were explored as a sacrificial scaffold for production of threads of ECM. Described herein are methods tasked for lab scale manufacturing of cell culture quality hollow fiber membranes and the extraction of bulk extracellular matrix fibers from the same following a period of fibroblast culture. This static culture approach is readily adoptable by laboratories containing standard mammalian cell culture equipment. ECM produced by this approach could be applied toward a variety of clinical applications.

<table><tbody><tr><td>1/32 inch thick silicone rubber</td><td>Grainger</td><td>B01LXJULOM</td><td></td></tr><tr><td>20 mL Scintillation Vials, Borosilicate glass, Disposable - VWR</td><td>VWR</td><td>66022-004</td><td>With attached white urea cap and cork foil liner</td></tr><tr><td>3 inch by 1 inch microscopy slides</td><td>VWR</td><td>75799-268</td><td></td></tr><tr><td>4C refrigerator</td><td>Thermo Fisher Scientific</td><td>FRGG2304D</td><td>Any commercial 4C refrigerator will suffice.</td></tr><tr><td>50 mL tubes</td><td>VWR</td><td>21008-178</td><td></td></tr><tr><td>6-well cell culture plates</td><td>VWR</td><td>10062-892</td><td>Alternative brands may be used</td></tr><tr><td>Acetone</td><td>VWR</td><td>E646</td><td>Alternative brands may be used</td></tr><tr><td>Bore vessel</td><td>McMaster-Carr</td><td>89785K867</td><td>6 ft 316 steel tubing</td></tr><tr><td>Bovine Plasma Fibronectin</td><td>Thermo Fisher Scientific</td><td>33010018</td><td>Comes as 1 mg of lyophilized protein</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>VWR/Amresco</td><td>97062-590</td><td></td></tr><tr><td>Cell Culture Incubator w/ CO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Any appropriate CO2-supplied mammalian cell incubator will suffice.</td></tr><tr><td>Disposable Serological Pipets, Glass - Kimble Chase</td><td>VWR</td><td>14673-208</td><td>Alternative brands may be used</td></tr><tr><td>DMEM/F-12, HEPES</td><td>Thermo Fisher Scientific</td><td>11330032</td><td>Warm in water bath at 37&amp;deg;C for 30 minutes prior to use</td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>DN25-10MG</td><td></td></tr><tr><td>Dope vessel</td><td>McMaster-Carr</td><td>89785K867</td><td>6 ft 316 steel tubing</td></tr><tr><td>Ethanol</td><td>VWR</td><td>BDH1160</td><td>Dilute to 70% for sterilization</td></tr><tr><td>Fetal Bovine Serum, qualified, US origin - Gibco</td><td>Thermo Fisher Scientific</td><td>26140079</td><td>Mix with growth media at 10% concentration (50mL in 500mL media)</td></tr><tr><td>Four 1/4-inch to 1&quot; reducing unions</td><td>Swagelok</td><td>SS-1610-6-4</td><td>One reducing union for each inlet and outlet of each vessel</td></tr><tr><td>Freeze-dryer/lyophilizer</td><td>Labconco</td><td>117 (A65312906)</td><td>Any lyophilizer will suffice.</td></tr><tr><td>Hexagonal Antistatic Polystyrene Weighing Dishes - VWR</td><td>VWR</td><td>89106-752</td><td>Any weigh boat will suffice</td></tr><tr><td>Hollow fiber membrane immersion bath</td><td></td><td></td><td>34L polypropylene tubs may be used or large bath containers can be fabricated from welded steel sheets</td></tr><tr><td>Hollow Fiber Membrane Spinneret</td><td>AEI</td><td>http://www.aei-spinnerets.com/specifications.html</td><td>Made to order. Inner diameter = 0.8 mm, outer diameter = 1.6 mm</td></tr><tr><td>Hot plate/stirrer</td><td>VWR</td><td>97042-634</td><td></td></tr><tr><td>Human TGF-&amp;beta;1</td><td>PeproTech</td><td>100-21</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4544-25G</td><td></td></tr><tr><td>L-Ascorbic acid 2-phosphate</td><td>Sigma-Aldrich</td><td>A8960-5G</td><td></td></tr><tr><td>L-glutamine (200 mM) - Gibco</td><td>Thermo Fisher Scientific</td><td>25030081</td><td>Mix with growth media at 1% concentration (5mL in 500mL media)</td></tr><tr><td>MgCl2</td><td>VWR/Alfa Aesar</td><td>AA12315-A1</td><td></td></tr><tr><td>Minus 80 Freezer</td><td>Thermo Fisher Scientific</td><td>UXF40086A</td><td>Any commercial -80C freezer will suffice.</td></tr><tr><td>N2 gas cylinders (two)</td><td></td><td></td><td></td></tr><tr><td>NIH/3T3 cells</td><td>ATCC</td><td>CRL-1658</td><td>Alternative fibrogenic cell lines may be used.</td></tr><tr><td>N-methyl-2-pyrrolidone</td><td>VWR</td><td>BDH1141</td><td>Alternative brands may be used</td></tr><tr><td>Penicillin/Streptomycin Solution - Gibco</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Mix with growth media at 0.1% concentration (0.5 mL in 500mL media)</td></tr><tr><td>Polysulfone</td><td>Sigma-Aldrich</td><td>428302</td><td>Any polysulfone with an average Mw of 35,000 daltons may be used</td></tr><tr><td>Portable Pipet-Aid Pipetting Device - Drummond</td><td>VWR</td><td>53498-103</td><td>Alternative brands may be used</td></tr><tr><td>PTFE tubing (1/4-inch inner diameter)</td><td>McMaster-Carr</td><td>52315K24</td><td>Alternative brands may be used.</td></tr><tr><td>Rat skeletal muscle fibroblasts</td><td></td><td></td><td>Independently isolated from rat skeletal muscle. Alternative fibrogenic cell lines may be used.</td></tr><tr><td>RNase A</td><td>Sigma-Aldrich</td><td>R4642</td><td></td></tr><tr><td>Silicone sheet</td><td>McMaster-Carr</td><td>1460N28</td><td></td></tr><tr><td>Take-up motor</td><td>Greartisan</td><td>B071GTTSV3</td><td>200 RPM DC Motor</td></tr><tr><td>Tris HCl</td><td>VWR/Amresco</td><td>97063-756</td><td></td></tr><tr><td>Two needle valves</td><td>Swagelok</td><td>SS-1RS4</td><td></td></tr></tbody></table>

production of extracellular matrix fibers via sacrificial hollow fiber membrane cell culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

Successful production of extracellular matrix from sacrificial scaffolds is contingent on appropriate scaffold fabrication, cell culture, and solvent rinse procedures. Fabrication of the hollow fiber membranes is performed using a dry-jet wet-spinning system assembled from commercially available components (Figure 1) which uses extrusion of polymer solution through the annulus of a commercially available steel spinneret (inner diameter = 0.8 mm, outer diamete...

Watch this Scientific Journal Video about Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture at JoVE.com

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

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7.4K Views.  University of Arkansas. The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes. 

Video: Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

Cancer Research

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Formation and Attachment of FN-Silk Membranes to Inserts of Cell Culture 

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

Author Spotlight: Enhancing In Vitro Cell Culture Models with Recombinant Functionalized Spider Silk Membranes

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...

Generation of Myocardium-Mimicking Extra Cellular Matrix for Cell Culture

Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. From the early stages of heart development throughout the lifetime, in both normal and pathological conditions, the composition of the extracellular matrix changes and influences myocardium structure and function. The purpose of the method described here is to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo. To this end, fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. The subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of the myocardium-specific extracellular matrix on other cells. Importantly, the composition of the fibroblast-derived coating of the culture dish changes according to the in vivo activity of the fibroblasts isolated from the heart, allowing subsequent studies of cell-matrix interactions in different normal and pathological conditions.

Author Spotlight: Studying Cardiac Cell-Matrix Interactions In Vitro

Fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. After decellularization, this substrate can be used for the culture and study of other cardiac cells and cell-matrix interactions.

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. ...

Biology

Histotypic Tissue Culture

Although two-dimensional tissue culture has been common for some time, cells behave more realistically in a three-dimensional culture, and more closely mimics native tissue. This video introduces histotypic tissue culture, where the growth and propagation of one cell line is done in an engineered three-dimensional matrix to reach high cell density. Here, we show the harvesting of cells from donor tissue, followed by cell culture on an engineered construct.

Histotypic Tissue Culture; 3D Culture on Scaffolds

Although two-dimensional tissue culture has been common for some time, cells behave more realistically in a three-dimensional culture, and more closely ...

Education

JoVE Science Education

Engineering

Bioscaffold Fabrication by Electrospraying: A Technique to Generate Microcarrier Scaffolds Derived from Extracellular Matrix of Decellularized Tissue

Source: Kornmuller, A. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/55436" target="_blank">Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms.</a> J. Vis. Exp. (2017)
This video demonstrates the procedure of extracellular matrix-derived microcarrier fabrication by electrospraying. These microcarriers simulate the native tissue-specific microenvironment and are stable in long-term 3D in vitro cell culture models.

Wytwarzanie rusztowań biologicznych przez elektronatryskiwanie: technika generowania rusztowań mikronośnikowych pochodzących z zewnątrzkomórkowej macierzy tkanki pozbawionej komórek

Source: Kornmuller, A. et al. Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms. ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

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Postproduction Processing of Electrospun Fibres for Tissue Engineering

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating

Histotypic Tissue Culture

Wytwarzanie rusztowań biologicznych przez elektronatryskiwanie: technika generowania rusztowań mikronośnikowych pochodzących z zewnątrzkomórkowej macierzy tkanki pozbawionej komórek