Name: silk film culture system for in vitro analysis and biomaterial design
Uploaded: 2012-04-24T12:00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Silk Film Culture System for in vitro Analysis and Biomaterial Design at JoVE.com

Funding from NIH K08EY015829, R21EY019561, R24EY015656, P41 EB002520, and R01 EY020856 Research to Prevent Blindness Career Development Award, and Tri-Institutional Stem Cell Initiative. HCLE cell line provided courtesy of Dr. Ilene Gipson. The authors would like to thank Dr. Aihong Liu at Weill Cornell Medical College for her technical assistance and guidance with cell culture, Anthony Labissiere at the Hospital for Special Surgeries for her technical assistance with SEM imaging, the Tissue Engineering Resource Center (TERC) at Tufts University for technical support with material development, and the Cornell Center for NanoScale Science and Technology Facility (CNF) for assistance in silicon wafer manufacturing.

1. Fabrication of Silicone Rubber Molds
<ol>
	<li>Produce or purchase a desired topographic surface for casting. For this publication a standard 100 mm etched silicon wafer will be described (Figure 1).</li>
	<li>Weigh out polydimethylsiloxane (PDMS) potting (component A) and catalyst (component B) solution in a 1:9 ratio (9 g potting and 1 g catalyst) as provided in the purchased kit.</li>
	<li>Mix solutions thoroughly to initiate curing process.</li>
	<li>Place silicon wafer surface within a casting dish.</li>
	<li>Weigh out 4.5 g of PDMS solution onto silicon wafer.</li>
	<li>Spread PDMS solution as to cover a 100 mm diameter area of the wafer surface.</li>
	<li>Tilt wafer to spread PDMS solution evenly.</li>
	<li>Cover wafer with 100 mm diameter petri dish lid.</li>
	<li>Place casting setup into 60 &deg;C oven over night, making certain that curing is taking place on a flat surface.</li>
	<li>Place cured PDMS/silicon wafer into 70% ethanol bath before removal.</li>
	<li>Begin removing PDMS from wafer by using razor blade to lift edge (entire circumference) first.</li>
	<li>Gently pull PDMS off using forceps within a 70% ethanol bath being careful not to tear silicone rubber casting.</li>
	<li>Punch out individual PDMS molds using 14 mm hole punch. This diameter is designed to fit into a 24-well plate setup.</li>
</ol>
2. Production of Silk Solution
<ol>
	<li>Bring 2 L of distilled water (dH2O) to a boil within a glass beaker 7 .</li>
	<li>Cut 5 g of Bombyx mori silkworm cocoons into thirds.</li>
	<li>Dispose of extensively contaminated cocoons as indicated by excessive insect particulates coating the inner cocoon surface.</li>
	<li>Measure 4.24 g of sodium carbonate.</li>
	<li>Add sodium carbonate slowly to boiling dH2O volume to prevent boiling over of water, and allow complete dissolution before continuing.</li>
	<li>Add cocoon pieces to boiling dH2O for 40 min., and use a Teflon coated stir bar to stir cocoons during boiling process.</li>
	<li>After boiling, carefully drain dH2O into sink and ring out the silk extract by hand to remove excess water.</li>
	<li>Wash silk extract three times for 20 min. each in 1 L of dH2O in a lab beaker, and use a stir bar to circulate volume within beaker.</li>
	<li>After washing, ring out the silk extract by hand and place silk fiber extract inside a chemical hood to allow for drying for a 12 hr. period.</li>
	<li>Next day, weigh the dried silk fibers, which is typically ~3.5 g: ___________-g</li>
	<li>Prepare 9.3 M LiBr solution for a 20% w/v solution of silk. Utilize following equations to calculate necessary weight and volumes:
	<ol class="lalpha">
		<li>(Silk extract weight from step 10) x (4) = ___________-mL of total 9.3 M LiBr solution</li>
		<li>[(807.705) x (LiBr volume from part 11.a)]/1000 = ___________ g of LiBr to add to dH2O</li>
	</ol>
	</li>
	<li>Add measured LiBr weight into a glass beaker of the following dH2O volume:
	<ol class="lalpha">
		<li>(0.8) x (calculated volume from step 11.a) = ___________ mL of dH2O</li>
	</ol>
	</li>
	<li>Pour this solution into an appropriate sized graduated cylinder, and bring solution up to final volume as calculated in part 11.a.</li>
	<li>Place the silk extract into beaker and pour LiBr solution over the silk fibers making sure the silk fibers are immersed within LiBr solution using a lab spatula.</li>
	<li>Place the dissolved silk into 60 &deg;C oven for 4 hr: 
	Start time: ___________ 
	End time: ___________</li>
	<li>Using an appropriate sized syringe draw up 12 mL of the silk solution. Place an 18G needle on end of the syringe, and then inject the solution into a dialysis casette (3,500 MW coutoff, Slide-A-Lyzer, Thermo Scientific). After filling the cassette, draw the remaining air out of the cassette with the emptied syringe.</li>
	<li>Place filled dialysis cassette within 1 L of dH2O.</li>
	<li>Change dH2O volume after 1 hr, 4 hr, 8 hr and then every 12 hr 3x for a total of 6 changes:
	<ol class="lalpha">
		<li>Begin: ___________</li>
		<li>1hr: ___________</li>
		<li>4hr: ___________</li>
		<li>8 hr: ___________</li>
		<li>12 hr: ___________</li>
		<li>12 hr: ___________</li>
		<li>12 hr: ___________</li>
		<li>End: ___________</li>
	</ol>
	</li>
	<li>After dialysis, slowly collect the silk solution from cassettes with syringe. Place solution into 10,000 g rated centrifuge tubes.</li>
	<li>Centrifuge the solution twice at 10,000 g at 4 &deg;C for 20-min each. After each centrifugation place supernatant into a new tube.</li>
	<li>Store thr silk solution in 4 &deg;C refrigerator.</li>
	<li>Pipette 2 samples of 0.5 mL silk solution into small weigh dish, and place weigh dishes inside a 60 &deg;C dry oven for 12 hours or until the silk solution dries.</li>
	<li>Weigh the remaining solid silk film from both samples to measure silk solution percent weight to volume protein concentration:
	<ol class="lalpha">
		<li>Weight of combined 2 silk film samples:___________ mg</li>
		<li>(Weight from 23.a) x (100) = ___________% silk</li>
	</ol>
	</li>
</ol>
3. Preparation of Silk Films and Culture System Setup
<ol>
	<li>Prepare PDMS casting surfaces by cleaning with clear tape to remove dust.</li>
	<li>Clean PDMS substrates by soaking in 70% EtOH and wash three times with dH2O.</li>
	<li>Place 14 mm PDMS molds onto holder plate, which is typically a 24 well plate lid.</li>
	<li>To produce a 50 &mu;m thick film, spread 70 &mu;ls of 8% silk solution on PDMS molds using a liquid spreading tool that is typically a 1 mL syringe tip 2.</li>
	<li>Allow the silk films to dry uncovered on a clean bench running an air flow pressure of 150 Pa for a period of 90 min or until films are dry.</li>
	<li>Once films are dry place entire set of films, including PDMS molds, into a water-annealing chamber for &gt;4 hrs to produce a water insoluble film. This is typically an empty desiccator chamber with water in the bottom of the basin pulled at a 25 kPa vacuum 15 .</li>
	<li>Remove silk films from water-annealing chamber and place onto a clean bench.</li>
	<li>Prepare a 70% EtOH bath within a 35 mm Petri dish, and place control substrates (i.e. glass or plastic surfaces) and stainless steel rings within wells for at least 10 min to sterilize.</li>
	<li>Remove silk films from PDMS molds using forceps, dip them into 70% EtOH, and place sample into 24-well plate prefilled with 1 mL of 70% EtOH making sure patterned side is facing up to allow for cell adhesion.</li>
	<li>After placing each silk film sample into a corresponding well place stainless steel ring weights (11.65 mm inner diameter, 15.45 mm outer diameter, and 1.18 mm thickness) on top.</li>
	<li>Allow samples with rings to incubate for 10 minutes to ensure sterility.</li>
	<li>Remove EtOH and wash each sample 3x with 1 mL of PBS. Let each wash sit for 5 min to allow for complete diffusion.</li>
	<li>Remove PBS using aspirating glass pipette, while making sure to remove majority of bubbles beneath silk film samples.</li>
	<li>Prepare cell line for seeding. As an example, trypsinize human corneal-limbal epithelial (HCLE) cell line with 0.25% trypsin and ethylenediaminetetraacetic acid (EDTA) solution for 7-min. Deactivate trypsin using 1:1 volume of Dulbecco's modified Eagle medium (DMEM) passage media with 10% FBS added. Centrifuge trypsinized HCLE cells, add 8-mL of keratinocyte-serum free media (K-SFM) to cell pellet, gently agitate to disperse HCLEs, and generate cell count.</li>
	<li>Sample 500 &mu;L of HCLE suspension per well typically using a 10,000 cells/cm2 density.</li>
	<li>Place cultures within incubator at 37 &deg;C and 5% CO2 and run desired experimental protocol.</li>
</ol>
4. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/3646/3646fig1.jpg" /> 
Figure 1. Flow chart illustrating summarized 10-step process of silk film production and culture system preparation.
<img alt="Figure 2" src="/files/ftp_upload/3646/3646fig2.jpg" /> 
Figure 2. (A) 21-dye array of patterned line surface topographies produced upon a 90 mm diameter silicon wafer employing standard photolithographic and dry etching techniques. (B) Silk films retain original parallel line patterned features after casting on PDMS molding surfaces. (C) Schematic demonstrating feature size design chosen to promote cellular alignment. (D) Cross-section of silk film illustrating retained parallel lined patterned surface.
<img alt="Figure 3" src="/files/ftp_upload/3646/3646fig3.jpg" /> 
Figure 3. Scanning electron micrographs of HCLE cell line adhering to (A) patterned and (B) flat silk films at day 2 in culture. HCLE cultures continue to proliferate to confluence on (C) patterned and (D) flat surfaces by day 8 in culture.
<img alt="Figure 4" src="/files/ftp_upload/3646/3646fig4.jpg" /> 
Figure 4. (A, D, G) Patterned and (B, E, H) flat silk films culture HCLE cells comparatively to (C, F, I) glass control substrates at (A-C) day 1, (D-F) day 4, and (G-I) day 8 in culture. (J) CyQuant nucleic acid content and (K) (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic activity assay data demonstrating HCLE viability on both patterned and flat silk film substrates when compared to glass control surfaces over time (scale bars = 100 &mu;m).
Video 1. Time lapse phase-contrast imaging of HCLE cells migrating over a patterned silk film surface during an 18 hr. time period. Cells were seeded at 10k/cm2 density and cultured for 2 hr. before imaging. <a href="https://www.jove.com/files/ftp_upload/3646/3646movie1.MOV" target="_blank">Click here to view movie</a>.
Video 2. Time lapse phase-contrast imaging of HCLE cells migrating over a flat TCP control surface during an 18 hr. time period. Cells were seeded at 10k/cm2 density and cultured for 2 hr. before imaging. <a href="https://www.jove.com/files/ftp_upload/3646/3646movie2.MOV" target="_blank">Click here to view movie</a>.

<ol>
	<li>Rockwood, D., Preda, R. C., Yucel, T., Wang, X., Lovett, M. L., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+Fabrication+from+Bombyx+mori+Silk+Fibroin.">Materials Fabrication from Bombyx mori Silk Fibroin.</a> Nature protocols. , (2011).</li><li>Lawrence, B., Omenetto, F., Chui, K., Kaplan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+methods+to+control+silk+fibroin+film+biomaterial+features.">Processing methods to control silk fibroin film biomaterial features.</a> Journal of materials science. 43, 6967-6985 (2008).</li><li>Altman, G., Diaz, F., Jakuba, C., Calabro, T., Horan, R., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk-based+biomaterials.">Silk-based biomaterials.</a> Biomaterials. 24, 401-416 (2003).</li><li>Hofmann, S., Wong, P. o., Foo, C., Rossetti, F., Textor, M., Vunjak-Novakovic, G., Kaplan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+fibroin+as+an+organic+polymer+for+controlled+drug+delivery.">Silk fibroin as an organic polymer for controlled drug delivery.</a> Journal of Controlled Release. 111, 219-227 (2006).</li><li>Demura, M., Asakura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+of+glucose+oxidase+with+Bombyx+mori+silk+fibroin+by+only+stretching+treatment+and+its+application+to+glucose+sensor.">Immobilization of glucose oxidase with Bombyx mori silk fibroin by only stretching treatment and its application to glucose sensor.</a> Biotechnology and bioengineering. 33, 598-603 (1989).</li><li>Cebe, P., Kaplan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+enzymatic+degradation+of+beta-sheet+crystals.">Mechanism of enzymatic degradation of beta-sheet crystals.</a> Biomaterials. , (2010).</li><li>Lawrence, B., Cronin-Golomb, M., Georgakoudi, I., Kaplan, D., Omenetto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+silk+protein+biomaterial+systems+for+optical+devices.">Bioactive silk protein biomaterial systems for optical devices.</a> Biomacromolecules. 9, 1214-1220 (2008).</li><li>Meinel, L., Hofmann, S., Karageorgiou, V., Kirker-Head, C., McCool, J., Gronowicz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+responses+to+silk+films+in+vitro+and+in+vivo.">The inflammatory responses to silk films in vitro and in vivo.</a> Biomaterials. 26, 147-155 (2005).</li><li>Vepari, C., Kaplan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+as+a+biomaterial.">Silk as a biomaterial.</a> Progress in Polymer Science. 32, 8-9 (2007).</li><li>Li, M., Ogiso, M., Minoura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+degradation+behavior+of+porous+silk+fibroin+sheets.">Enzymatic degradation behavior of porous silk fibroin sheets.</a> Biomaterials. 24, 357-365 (2003).</li><li>Arai, T., Freddi, G., Innocenti, R., Tsukada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradation+of+Bombyx+mori+silk+fibroin+fibers+and+films.">Biodegradation of Bombyx mori silk fibroin fibers and films.</a> Journal of Applied Polymer Science. 91, 2383-2390 (2004).</li><li>Lawrence, B., Marchant, J., Pindrus, M., Omenetto, F., Kaplan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+film+biomaterials+for+cornea+tissue+engineering.">Silk film biomaterials for cornea tissue engineering.</a> Biomaterials. 30, 1299-1308 (2009).</li><li>Ma, X., Cao, C., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+of+silk+fibroin+films+containing+sulfonated+silk+fibroin.">The biocompatibility of silk fibroin films containing sulfonated silk fibroin.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. 78, 89-96 (2006).</li><li>Patel, A., Thakar, R., Chown, M., Ayala, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+mechanisms+of+single-cell+interactions+with+microtopographical+cues.">Biophysical mechanisms of single-cell interactions with microtopographical cues.</a> Biomedical. , (2010).</li><li>Jin, H., Park, J., Karageorgiou, V., Kim, U., Valluzzi, R., Cebe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-Stable+Silk+Films+with+Reduced+&#x03B2;-Sheet+Content.">Water-Stable Silk Films with Reduced &#x03B2;-Sheet Content.</a> Advanced Functional Materials. 15, 1241-1247 (2005).</li><li>Lawrence, B., Wharram, S., Kluge, J., Leisk, G., Omenetto, F., Rosenblatt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Hydration+on+Silk+Film+Material+Properties.">Effect of Hydration on Silk Film Material Properties.</a> Macromolecular Bioscience. 10, 393-403 (2010).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+silk+fibroin+as+a+support+for+enzyme+immobilization.">Natural silk fibroin as a support for enzyme immobilization.</a> Biotechnology Advances. 16, 961-971 (1998).</li><li>Cebe, P., Kaplan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+enzymatic+degradation+of+beta-sheet+crystals.">Mechanism of enzymatic degradation of beta-sheet crystals.</a> Biomaterials. , (2010).</li><li>Shaw, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+the+fibroin+of+Bombyx+mori+with+trypsin.">Fractionation of the fibroin of Bombyx mori with trypsin.</a> Biochemical Journal. 93, 45 (1964).</li><li>Rice, W., Firdous, S., Gupta, S., Hunter, M., Foo, C., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+characterization+of+structure+and+morphology+of+silk+fibroin+biomaterials+using+non-linear+microscopy.">Non-invasive characterization of structure and morphology of silk fibroin biomaterials using non-linear microscopy.</a> Biomaterials. 29, 2015-2024 (2008).</li><li>Chirila, T., Barnard, Z., Harkin, D., Schwab, I., Hirst, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bombyx+mori+silk+fibroin+membranes+as+potential+substrata+for+epithelial+constructs+used+in+the+management+of+ocular+surface+disorders.">Bombyx mori silk fibroin membranes as potential substrata for epithelial constructs used in the management of ocular surface disorders.</a> Tissue Engineering Part A. 14, 1203-1211 (2008).</li><li>Hu, X., Shmelev, K., Sun, L., Gil, E. -. S., Park, S. -. H., Cebe, P. <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+Silk+Material+Structure+by+Temperature-Controlled+Water+Vapor+Annealing.">Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing.</a> Biomacromolecules. 12, 1686-1696 (2011).</li><li>Omenetto, F., Kaplan, 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+new+route+for+silk.">A new route for silk.</a> Nature Photonics. 2, 641-643 (2008).</li><li>Bray, L. J., George, K. A., Ainscough, S. L., Hutmacher, D. W., Chirila, T. V., Harkin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+epithelial+equivalents+constructed+on+Bombyx+mori+silk+fibroin+membranes.">Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes.</a> Biomaterials. 32, 5086-5091 (2011).</li><li>Wang, N., Tytell, J. D., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+at+a+distance:+mechanically+coupling+the+extracellular+matrix+with+the+nucleus.">Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus.</a> Nat. Rev. Mol. Cell Biol. 10, 75-82 (2009).</li><li>Jaalouk, D. E., Lammerding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+gone+awry.">Mechanotransduction gone awry.</a> Nat. Rev. Mol. Cell Biol. 10, 63-73 (2009).</li></ol>

No conflicts of interest declared.

The use of regenerated silk films as a substrate for cell culture has gained in popularity over the past two decades due to extensive characterization of the material properties of this protein and increased understanding of its biomaterial utility 3,8 . The culture system described here represents a novel in vitro testing system for assessing cell surface interactions on patterned silk film biomaterial substrates 7 . The system allows for in depth analysis of cellular interactions over time that can be easily adopted for high-throughput data collection. This is largely enabled because silk films possess a number of tunable biomaterial properties that can be modified to directly affect cell function 8,9,12, including: control of surface micro/nano-surface topography 7; various surface chemistries through covalent modification or adsorption of biologically active molecules 13; robust mechanical properties 15,16; control of material hydrophilicity/hydrophobicity 16 ; bulk loading of biological compounds for release 4,8,17 ; and controlled dissolution/enzymatic degradation rates through control of the secondary structure (beta sheet content) 11,18,19 .
The transparency of silk films is achieved through annealing the films for a period of time under vacuum in the presence of water vapor 7,15. This processing approach allows for the formation of &beta;-sheet secondary structure, fostering the insolubility of the material in water while allowing minimal diffraction of light 15. This transparency of films is key in enabling direct live-cell imaging that can be employed under a number of imaging modalities (i.e. wide-field and fluorescence) using any number of microscope systems 12,20. In addition to live-cell imaging, silk films can be easily removed from the culture system to allow for additional fixation and analysis. Thus, the large variety of direct experimental assessments that can be performed on this system are applicable to a wide variety of cell/tissue sources for many technical fields 3,8,9,12,13,21. The time-lapse imaging results illustrate how real-time culture data can be collected, and as an example were utilized to illustrate how surface topography affects cell interactions. The representative results demonstrate how silk film biomaterials can be utilized to support HCLE culture growth, and are amendable to a number of standard cellular proliferation and metabolic assays (Fig. 4). In addition, the cultures are fixed and processed for scanning electron imaging or other protocols (Fig. 3).
Silk film substrates are produced in the lab with high fidelity, consistency, and with relatively low cost (Fig. 1). This enables reproducibility in both culture system setup and experimental outcomes. It has been demonstrated that water-anneal processing produces a stable silk film material within culture that has defined degradation rates pending the concentration of proteases in solution 2,15,22. As a result these materials may be used for extended periods of time for long-term cell culture, or remain implanted for months or years depending on the physiological location 8. In addition, recent work has shown that both the protein structure and material properties of water-annealed silk films are consistent from batch to batch allowing for reproducible culture results as shown through various mechanical and biophysical testing methods 15,16. In addition, the material's surface has shown great fidelity amongst film batches as indicated by SEM, atomic force micrscopy (AFM), and cell culture studies {Lawrence:2008wr, Omenetto:2008tc, Bray:2011kq}7,23,24. Material stability and consistency is an important factor to how the cell will sense the culture substrate through the various mechanotransduction pathways, and ultimately produce a desired/undesired cellular response 25,26.
Historical standards for culture substrates, such as tissue culture treated plastic or glass, provide adequate substrates for cell attachment. However, these materials are not amendable for further utility in vivo. It can be envisioned that a silk film biomaterial could be customized in vitro, and once experimental expectations have been achieved the customized film can be directly translated to an in vivo model. Such paired design between in vitro and in vivo experimentation offers a great advantage for such implantable silk biomaterials over other substrates that are routinely used in vitro.

<table border="1">
<tbody>
 <tr>
 <td>Material Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Silk cocoons</td>
 <td>Tajima Shoji Co., LTD.</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>PDMS monomer and cross-linker</td>
 <td>Momentive</td>
 <td>RTV615A 01P</td>
 </tr>
 <tr>
 <td>Sodium Carbonate</td>
 <td>Sigma</td>
 <td>S2127</td>
 </tr>
 <tr>
 <td>Lithium Bromide</td>
 <td>Sigma</td>
 <td>213225</td>
 </tr>
 <tr>
 <td>Slide-A-Lyzer</td>
 <td>Thermo Scientific</td>
 <td>66110</td>
 </tr>
 <tr>
 <td>1 mL Syringe</td>
 <td>Becton-Dickenson</td>
 <td>309602</td>
 </tr>
 <tr>
 <td>Stainless steel washer</td>
 <td>Superior Washer</td>
 <td>81610</td>
 </tr>
 <tr>
 <td>24-well plate</td>
 <td>VWR</td>
 <td>353047</td>
 </tr>
</tbody>
</table>

silk film culture system for in vitro analysis and biomaterial design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Watch this Scientific Journal Video about Silk Film Culture System for in vitro Analysis and Biomaterial Design at JoVE.com

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

silk-film-culture-system-for-in-vitro-analysis-and-biomaterial-design

Research

JoVE Journal

Bioengineering

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

Design and Analysis for Fall Detection System Simplification

This paper presents a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition system that can be easily implemented and adopted. The methodology is based on the configuration of specific types of sensors, machine-learning methods and procedures. The protocol is divided into four phases: (1) database creation (2) data analysis (3) system simplification and (4) evaluation. Using this methodology, we created a multimodal database for fall detection and human activity recognition, namely UP-Fall Detection. It comprises data samples from 17 subjects that perform 5 types of falls and 6 different simple activities, during 3 trials. All information was gathered using 5 wearable sensors (tri-axis accelerometer, gyroscope and light intensity), 1 electroencephalograph helmet, 6 infrared sensors as ambient sensors, and 2 cameras in lateral and front viewpoints. The proposed novel methodology adds some important stages to perform a deep analysis of the following design issues in order to simplify a fall detection system: a) select which sensors or combination of sensors are to be used in a simple fall detection system, b) determine the best placement of the sources of information, and c) select the most suitable machine learning classification method for fall and human activity detection and recognition. Even though some multimodal approaches reported in literature only focus on one or two of the above-mentioned issues, our methodology allows simultaneously solving these three design problems related to a human fall and activity detection and recognition system.

We present a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition system. The goal is to build a system for accurate fall detection that can be easily implemented and adopted.

This paper presents a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition ...

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 ...