Name: production of extracellular matrix fibers via sacrificial hollow fiber membrane cell culture
Uploaded: 2019-02-02T13:00:00
Description: Watch this Scientific Journal Video about Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture at JoVE.com

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 po...

<ol>
	<li>Cobb, W. S., Kercher, K. W., Heniford, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+argument+for+lightweight+polypropylene+mesh+in+hernia+repair.">The argument for lightweight polypropylene mesh in hernia repair.</a> Surgical Innovation. 12 (1), 63-69 (2005).</li><li>Morais, J. M., Papadimitrakopoulos, F., Burgess, D. 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. C., Tresco, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+cell+derived+extracellular+matrix+constructs+using+sacrificial+open-cell+foams.">The isolation of cell derived extracellular matrix constructs using sacrificial open-cell foams.</a> Biomaterials. 31 (36), 9595-9603 (2010).</li><li>Roberts, K., Schluns, J., Walker, A., Jones, J. D., Quinn, K. P., Hestekin, J., Wolchok, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+derived+extracellular+matrix+fibers+synthesized+using+sacrificial+hollow+fiber+membranes.">Cell derived extracellular matrix fibers synthesized using sacrificial hollow fiber membranes.</a> Biomedical Materials. 13 (1), (2017).</li><li>Hurd, S. A., Bhatti, N. M., Walker, A. M., Kasukonis, B. M., Wolchok, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+biological+scaffold+engineered+using+the+extracellular+matrix+secreted+by+skeletal+muscle+cells.">Development of a biological scaffold engineered using the extracellular matrix secreted by skeletal muscle cells.</a> Biomaterials. 49, 9-17 (2015).</li><li>Kasukonis, B. M., Kim, J. T., Washington, T. A., Wolchok, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+infusion+bioreactor+for+the+accelerated+preparation+of+decellularized+skeletal+muscle+scaffolds.">Development of an infusion bioreactor for the accelerated preparation of decellularized skeletal muscle scaffolds.</a> Biotechnology Progress. 32 (3), 745-755 (2016).</li><li>Murad, S., 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=Regulation+of+collagen+synthesis+by+ascorbic+acid.">Regulation of collagen synthesis by ascorbic acid.</a> Proceedings of the National Academy of Sciences of the United States of America. 78 (5), 2879-2882 (1981).</li><li>Zhang, Y., 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=Tissue-specific+extracellular+matrix+coatings+for+the+promotion+of+cell+proliferation+and+maintenance+of+cell+phenotype.">Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype.</a> Biomaterials. 30 (23-24), 4021-4028 (2009).</li><li>Feng, C. Y., Khulbe, K. C., Matsuura, T., Ismail, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progresses+in+polymeric+hollow+fiber+membrane+preparation,+characterization+and+applications.">Recent progresses in polymeric hollow fiber membrane preparation, characterization and applications.</a> Separation and Purification Technology. 111, 43-71 (2013).</li><li>Domb, A. J., Kost, J., Wiseman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biodegradable Polymers. , (1998).</li></ol>

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>CaCl2</td>
			<td>VWR/Amresco</td>
			<td>97062-590</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Incubator w/ CO2</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&#176;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&#34; 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-&#946;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 ...

This protocol allows for the production of filaments of bulk extracellular matrix from cultured cell lines, allowing for research into cell matrix interactions, as well as research into the potential of this biomaterial for wound repair. These synthetic fibers can avoid the foreign body response directed against synthetic materials, while still drawing from the rich repertoire of textile fabrication techniques to create a wide range of woven implantables. Demonstrating the procedure will be Cassandra Reed, a graduate student from my lab.Before seeding the cells, autoclave the hollow fiber membranes at 121 degrees Celsius for 30 minutes, followed by treatment with 50 milliliters of bovine plasma fibronectin in PBS at 37 degrees Celsius in 5%carbon dioxide for one hour. At the end of the incubation, use sterile micro scissors to cut the fibers to six centimeters or less, and use a one milliliter syringe equipped with a 21 gauge needle to seed one times 10 to the fifth fibroblast cells directly into the lumina of the hollow fiber membranes. Place six seeded fibers into a six inch diameter Petri dish, and briefly incubate the seeded fibers at 37 degrees Celsius in 5%carbon dioxide.After five minutes, transfer the fibers from the incubator into a new six centimeter Petri dish containing fibroblast culture medium supplemented with L-ascorbic acid, L-ascorbic acid 2 phosphate, and transforming growth factor beta 1 for up to three weeks in the cell culture incubator. At the end of the incubation, use forceps o transfer the cultured fibers into individual scintillation vials, and tilt each vial to allow the addition of up to five milliliters of N-Methyl-2-pyrrolidone down the side of the vials. Then use a serological pipette to slowly aspirate the N-Methyl-2-pyrrolidone from each fiber, and transfer the fibers into new scintillation vials for a second immersion in fresh N-Methyl-2-pyrrolidone.After the third immersion, rinse the resulting extracellular matrix threads three times in deionized water in a similar manner, and place a three-inch-long, one-inch wide 1/23rd-inch-thick piece of silicone rubber with a central eight-by-four millimeter rectangular mold onto a standard three-by-one inch glass microscope slide. After autoclaving, lay each extracellular matrix fiber side-by-side in the eight-by-four millimeter silicone mold until there is no visible open space. Place the mold into a 50 milliliter conical tube, and freeze the fibers at negative 80 degrees Celsius until completely frozen.For decellularization, incubate the mold in 1%sodium dodecyl sulfate for 24 hours at room temperature on a rocker with gentle agitation. The next day, rinse the extracted extracellular matrix with three washes in three milliliters of PBS per wash, and fill the mold with freshly prepared DNAs/RNAs digestion buffer for a six hour incubation at four degrees Celsius. At the end of the digestion, aspirate the digestion solution and rinse the mold three times in sterile PBS as demonstrated.After the third wash, incubate the scaffolds in 10%Penicillin-Streptomycin in PBS at four degrees Celsius overnight before freezing in a 50 milliliter conical tube at negative 80 degrees Celsius for about one hour. When the mesh has completely frozen, lyophilize the decellularized extracellular mesh overnight, or until completely dry, and transfer the lyophilized scaffolds to a sterile container within a bio safety hood and store at four degrees Celsius until use. Here a transverse cross-section of a polysulfone hollow fiber membrane fabricated using this protocol is shown, exhibiting outer and inner layers of finger-like pores characteristic of an asymmetric membrane.After fibroblast cell seeding and culture, N-Methyl-2-pyrrolidone rinsing as demonstrated, translucent threads of extracellular matrix are produced. The extracellular matrix producing cells remain viable inside the hollow fiber membranes throughout the entire three week culture period. The extracted matrix has a translucent appearance when hydrated, exhibiting an off-white color and fibrous appearance with a gross longitudinal alignment upon mesh assembly and lyophilization.The most critical step is the dissolving process of the cultured hollow fiber membranes in the N-Methyl-2-pyrrolidone solvent in order to extract the extracellular matrix. The material produced by this method can be used for fabricating tissue engineered implants for investigating the repair of tissues in pre-clinical studies. The N-Methyl-2-pyrrolidone used in this protocol is a skin and eye irritant, and therefore it is recommended that you handle the chemical using nitrile gloves, goggles, a lab coat, and handle it within a fume hood.

production-extracellular-matrix-fibers-via-sacrificial-hollow-fiber

Research

JoVE Journal

Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

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 ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

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 ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

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

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

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

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

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...