We are grateful to Dr. K. Sekiguchi (Institute for Protein Research, Osaka University), Dr. M. Yamada (Institute for Protein Research, Osaka University), Dr. T. Ikejima (China-Japan Research Institute of Medical and Pharmaceutical Sciences, Wuya College of Innovation, Shenyang Pharmaceutical University) and T. Hayashi (China-Japan Research Institute of Medical and Pharmaceutical Sciences, Wuya College of Innovation, Shenyang Pharmaceutical University) for helpful comments.

1. Preparation of keratinocyte culture medium
NOTE: Perform all procedures under aseptic conditions.
<ol>
	<li>Add 5 mL of penicillin-streptomycin and 10 mL of additive supplement K-1 to 500 mL of K110 Type-II medium (K110) using a pipette.</li>
</ol>
2. Preparation of the fibril form of type I collagen
NOTE: Perform procedures until step 2.6 under aseptic conditions.
<ol>
	<li>Keep 10x phosphate-buffered saline (PBS (-)), deionized water, collagen, a 96-well culture plate, and an empty 2-mL tube on ice. 
	NOTE: Do not use the same plate to prepare the fibril and non-fibrous forms.</li>
	<li>Add 1.12 mL of deionized water to an empty 2 mL tube using a pipette. Add 200 &#181;L of 10x PBS (-) to the deionized water in the 2 mL tube using a pipette. Gently shake the tube several times.</li>
	<li>Add 0.66 mL of collagen to the 2 mL tube using a pipette. Gently and quickly shake the tube several times. 
	NOTE: Avoid bubble formation in the solution as much as possible. Prepare collagen solution immediately prior to use.</li>
	<li>Pour 100 &#181;L of the collagen solution from step 2.3 into each well of the 96-well culture plate. Gently shake the culture plate in a left to right motion. 
	NOTE: Cover the entire surface of the wells with the collagen solution. If the solution does not cover the entire surface, spread the solution using a pipette tip. Avoid bubble formation in the solution as much as possible.</li>
	<li>Place the culture plate in a CO2 incubator and incubate at 37 &#176;C for 1 h. Check the gelation of collagen and move the culture plate to a clean bench. 
	NOTE: Confirm gelation by tilting the&#160;culture plate.</li>
	<li>Gently pour 150 &#181;L of K110 on the gels using a pipette along the wall of the well, place the culture plate in a CO2 incubator and incubate at 37 &#176;C for 1 h. Move the culture plate to a clean bench. Prior to cell culture, gently discard the K110 using a pipette. 
	NOTE: To protect the gels, the tip of the pipette should touch the wall of the well.</li>
</ol>
3. Preparation of the non-fibrous form of type I collagen
NOTE: Perform all procedures under aseptic conditions.
<ol>
	<li>Add 4 &#181;L of collagen to 1.2 mL of 1 mM hydrochloric acid (HCl) in a 1.5 mL tube and gently mix using a pipette. 
	NOTE: Prepare collagen immediately prior to use. Keep collagen and HCl chilled until the time of use.</li>
	<li>Pour 100 &#181;L of collagen into each well of a 96-well culture plate using a pipette. Gently shake the culture plate in a left to right motion and incubate at room temperature for 1 h. 
	NOTE: Ensure that the entire surface of the wells is covered with solution.</li>
	<li>After the incubation, discard the collagen solution and wash the wells with PBS (-) twice using a pipette.</li>
	<li>Pour 150 &#181;L of 1% bovine serum albumin/PBS (-) (1% BSA) to a well of the 96-well culture plate and incubate at room temperature for 1 h. Prior to cell culture, discard the 1% BSA. 
	NOTE: Ensure that the entire surface of the wells is covered with solution.</li>
</ol>
4. Culture of FEPE1L-8 cells
NOTE: Perform all procedures under aseptic conditions.
<ol>
	<li>Maintain FEPE1L-8 cells in K110 in a 100 mm culture dish in a CO2 incubator and incubated at 37 &#176;C and 5% CO2, to semi-confluency.</li>
	<li>Prepare K110, trypsin, and trypsin inhibitor in a water bath at 37 &#176;C.</li>
	<li>Carefully remove the medium from the culture dish, add 3 mL of 0.05% trypsin using a pipette, place the dish in a CO2 incubator and incubate at 37 &#176;C for 5 min. After incubation, check cell detachment from the surface of the culture dish using phase-contrast microscopy at 10x magnification. 
	NOTE: The morphology of the detached cells becomes round. If the cells spread, incubate for an additional period of 5 min.</li>
	<li>Add 3 mL of trypsin inhibitor and collect the detached cells in a 15 mL centrifuge tube using a pipette. Centrifuge the cells in a 15 mL centrifuge tube at 200 x g for 5 min.</li>
	<li>Discard the supernatant and re-suspend the pellet in 10 mL of K110 using a pipette. Count the cells using phase-contrast microscopy at 10x magnification and prepare cell concentration at 5.0 x 104&#160;cells/mL by appropriate dilution with K110.</li>
	<li>Gently seed 0.1 mL of K110 with cells in each well of the culture plate using a pipette along the wall of the well. Place the culture plate in a CO2 incubator and incubate at 37 &#176;C for the indicated time (2 h, 1 day, 3 days). 
	NOTE: To protect the gels, the tip of the pipette should touch the wall of the well.</li>
</ol>
5. Estimation of the number of viable cells
<ol>
	<li>Incubate K110 in a water bath at 37 &#176;C. Mix 130 &#181;L of tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H&#160;tetrazolium, monosodium salt (WST-8), and 1.3 mL of K110 in a 2-mL tube using a pipette.</li>
	<li>Move the culture plate to a clean bench and gently discard the K110 using a pipette. Gently wash away non-adherent cells with K110 by using a pipette</li>
	<li>Add 110 &#181;L of K110 mixed with WST-8 in each well using a pipette, place the culture plate in a CO2 incubator and incubate at 37 &#176;C for 2 h.</li>
	<li>Move the culture plate to a clean bench, collect 100 &#181;L of the conditioned medium from each well, and move it into a well of another 96-well culture plate. Measure absorbance at a wavelength of 450 nm (OD450) using a microplate reader and estimate the number of viable cells.</li>
</ol>

<ol>
	<li>Kanta, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+as+a+tool+in+studying+fibroblastic+cell+behavior.">Collagen as a tool in studying fibroblastic cell behavior.</a> Cell Adhesion &amp; Migration. 9 (4), 308-316 (2015).</li><li>Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+adhesion+to+collagen+substrates.">Fibroblast adhesion to collagen substrates.</a> Methods of Enzymology. 82, 503-508 (1982).</li><li>Kleinman, H. K., Haralsonand, M. A., Hassell, J. R., 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=Use+of+extracellular+matrix+and+its+components+in+culture.">Use of extracellular matrix and its components in culture.</a> Extracellular Matrix: A Practical Approach, 1st ed. , 289-301 (1995).</li><li>Yamato, M., Hayashi, T., Ninomiya, Y., Olsen, B. R., Ooyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topological+distribution+of+collagen+binding+sites+on+fibroblasts+cultured+within+collagen+gels.">Topological distribution of collagen binding sites on fibroblasts cultured within collagen gels.</a> Extracellular Matrix-Cell Interaction. , 123-140 (1998).</li><li>Suzuki, Y., Someki, I., Adachi, E., Irie, S., Hattori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+collagen+molecules+from+the+aspect+of+fibril+formation:+Acid-soluble,+alkali-treated+and+MMP1-digested+fragments+of+type+I+collagen.">Interaction of collagen molecules from the aspect of fibril formation: Acid-soluble, alkali-treated and MMP1-digested fragments of type I collagen.</a> Journal of Biochemistry. 126, 54-67 (1999).</li><li>Fujisaki, H., Hattori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+apoptosis+on+type+I+collagen+gel+caused+by+lack+of+laminin+5/10/11+deposition+and+Akt+signaling.">Keratinocyte apoptosis on type I collagen gel caused by lack of laminin 5/10/11 deposition and Akt signaling.</a> Experimental Cell Research. 280, 255-269 (2002).</li><li>Sasaki, J., Fujisaki, H., Adachi, E., Irie, S., Hattori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delay+of+cell+cycle+progression+and+induction+of+death+of+cancer+cells+on+type+I+collagen+fibrils.">Delay of cell cycle progression and induction of death of cancer cells on type I collagen fibrils.</a> Connective Tissue Research. 52 (3), 167-177 (2011).</li><li>Wang, Z., Li, R., Zhong, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+promotes+proliferation,+migration+and+airway+smooth+muscle+cells+in+a+rat+model+of+chronic+obstructive+pulmonary+disease+via+upregulation+of+the+PI3K/AKT+signaling+pathway.">Extracellular matrix promotes proliferation, migration and airway smooth muscle cells in a rat model of chronic obstructive pulmonary disease via upregulation of the PI3K/AKT signaling pathway.</a> Molecular Medicine Reports. 18, 3143-3152 (2018).</li><li>Shih, Y. -. R., Tseng, K. F., Lai, H. -. Y., Lin, C. -. H., Lee, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+stiffness+regulation+of+integrin&#8208;mediated+mechanotransduction+during+osteogenic+differentiation+of+human+mesenchymal+stem+cells.">Matrix stiffness regulation of integrin&#8208;mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells.</a> Journal of Bone and Mineral Research. 26, 730-738 (2011).</li><li>Breitkreutz, D., Koxholt, I., Thiemann, K., Nischt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+basement+membrane:+The+foundation+of+epidermal+integrity-+BM+functions+and+diverse+roles+of+binding+molecules+nidogen+and+perlecan.">Skin basement membrane: The foundation of epidermal integrity- BM functions and diverse roles of binding molecules nidogen and perlecan.</a> BioMed Resesrch International. , 179784 (2013).</li><li>Fujisaki, H., 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=Respective+optimal+calcium+concentrations+for+proliferation+on+type+I+collagen+fibrils+in+two+keratinocyte+line+cells,+HaCaT+and+FEPE1L-8.">Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8.</a> Regenerative Therapy. 8, 73-79 (2018).</li><li>Kaur, P., McDougall, J. K., Cone, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+primary+human+epithelial+cells+by+cloned+cervical+carcinoma+DNA+containing+human+papillomavirus+type+16+E6/E7+open+reading+frames.">Immortalization of primary human epithelial cells by cloned cervical carcinoma DNA containing human papillomavirus type 16 E6/E7 open reading frames.</a> Journal of General Virology. 70, 1261-1266 (1989).</li><li>Kaur, P., Carter, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+expression+and+differentiation+in+transformed+human+epidermal+cells+is+regulated+by+fibroblasts.">Integrin expression and differentiation in transformed human epidermal cells is regulated by fibroblasts.</a> Journal of Cell Science. 103, 755-763 (1992).</li><li>Fujisaki, H., Adachi, E., Hattori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+differentiation+and+proliferation+are+regulated+by+adhesion+to+the+three-dimensional+meshwork+structure+of+type+IV+collagen.">Keratinocyte differentiation and proliferation are regulated by adhesion to the three-dimensional meshwork structure of type IV collagen.</a> Connective Tissue Research. 49 (6), 426-436 (2008).</li><li>Hirose, M., Kosugi, H., Nakazato, K., Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+to+a+quiescent+and+contractile+phenotype+from+a+proliferative+phenotype+of+myofibroblast-like+human+aortic+smooth+muscle+cells+by+culture+on+type+IV+collagen+gels.">Restoration to a quiescent and contractile phenotype from a proliferative phenotype of myofibroblast-like human aortic smooth muscle cells by culture on type IV collagen gels.</a> Journal of Biochemistry. 125, 991-1000 (1999).</li><li>Yonemura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Sensitivity+of+Epithelial+Cells+to+Extracellular+Matrix+in+Polarity+Establishment.">Differential Sensitivity of Epithelial Cells to Extracellular Matrix in Polarity Establishment.</a> Plos One. 9, 112922 (2014).</li><li>Sato, K., 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=Possible+Involvement+of+Aminotelopeptide+in+Self-assembly+and+Thermal+Stability+of+Collagen+I+as+Revealed+by+Its+Removal+with+Proteases.">Possible Involvement of Aminotelopeptide in Self-assembly and Thermal Stability of Collagen I as Revealed by Its Removal with Proteases.</a> Journal of Biological Chemistry. 275 (33), 25870-25875 (2000).</li><li>Nakazato, K., Muraoka, M., Adachi, E., Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelation+of+lens+capsule+type+IV+collagen+solution+at+a+neutral+pH.">Gelation of lens capsule type IV collagen solution at a neutral pH.</a> Journal of Biochemistry. 120, 889-894 (1996).</li><li>Hattori, 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=Type+I+-Type+IV+collagen+hybrid+gel.">Type I -Type IV collagen hybrid gel.</a> European patent. , (2015).</li><li>Henriet, P., Zhong, Z. D., Brooks, P. C., Weinberg, K. I., DeClerck, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+with+fibrillar+collagen+inhibits+melanoma+cell+proliferation+by+up-regulating+p27KIP1.">Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (18), 10026-10031 (2000).</li><li>Liu, X., 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=Differential+levels+of+reactive+oxygen+species+in+murine+preadipocyte+3T3-L1+cells+cultured+on+type+I+collagen+molecule-coated+and+gel-covered+dishes+exert+opposite+effects+on+NF-&#954;B-mediated+proliferation+and+migration.">Differential levels of reactive oxygen species in murine preadipocyte 3T3-L1 cells cultured on type I collagen molecule-coated and gel-covered dishes exert opposite effects on NF-&#954;B-mediated proliferation and migration.</a> Free Radical Research. 28, 1-16 (2018).</li></ol>

The authors declare that they have no competing financial interests.

Some ECM components, including type I collagen, form three-dimensional structures in vivo1. Culturing on such a three-dimensional, gel substrate provides more physiological conditions in vitro than on a two-dimensional, plastic surface1,2,3,4. Numerous protocols regarding the gel culture method have been reported, such as using type I collagen1,2,3,4,6,7,11, type IV collagen14,15, and Matrigel16. Type I collagen is a well-defined and widely used material because of its abundance and ease of handling. The features of purified type I collagen depend on the animal species, age, and purification methods5,17. Type I collagen can be purified using acetic acid and/or proteases, such as pepsin, papain, and proctase5,17. Acid-soluble collagen maintains amino-telopeptides and protease-soluble collagen are cleaved amino-telopeptides5,17. The reserved length of amino-telopeptides depends on the type of proteases, and the presence of telopeptides affects fibril morphology and gel strength5,17. The viscosity of acid-soluble collagen fibrils is greater than that of protease-treated collagens17. In this study, we used acid-soluble bovine type I collagen. Pepsin-solubilized collagen can also be used in this gel culture protocol; however, the gel strength is weaker17. These differences in materials can cause cell behavioral differences, but they are currently not well understood.
The gel culture protocol described in this study is very simple. Many modifications of this method have been reported. One possible modification for the culture of keratinocytes is to mimic the basement membrane. Type IV collagen gels may be better to keep a basement membrane-like substrate structure in vitro15,18. However, a long incubation period is required for the preparation of type IV collagen gels14,15,18. Instead, mixing type IV collagen with type I collagen gels can produce novel culture substrates (type I/type IV collagen hybrid gels)19. These hybrid gels are easy to handle, require a short time for gelation, and yield more basement membrane-like conditions for keratinocytes. On type I/type IV collagen hybrid gels, keratinocytes survive, form colonies, and induce terminal differentiation19. This hybrid method has versatile applications.
On-gel culture using type I collagen affects cancer cells. On the fibril form of type I collagen, Akt activation and growth of Caco-2 cells (a colon cancer cell line) are suppressed7. In addition, the growth of human melanoma cells (M24met) on the fibril form is arrested at the G1/S checkpoint20. Moreover, markedly increased levels of reactive oxygen species are observed in murine 3T3-L1 preadipocytes cultured on the fibril form. Furthermore, cell proliferation and migration are stimulated in opposite directions by the non-fibrous and fibril forms of type I collagen21.

Interstitial connective tissues comprise a three-dimensional protein meshwork of heterogeneous composition, primarily composed of type I collagen fibrils1. Collagen fibrils play a key role as a scaffold for cells1,2,3 and interact with other extracellular matrix (ECM) proteins3. In vitro, different forms of type I collagen can be used as substrates for cell culture depending on the handling method1,2,3,4. Under acidic conditions, type I collagen maintains the non-fibrous form5. Coating the surface of culture dishes with the non-fibrous form promotes cell adhesion and proliferation6,7. At physiological pH and temperature, type I collagen molecules reassemble into fibrils that form gels possessing a three-dimensional structure1,2,3,4,5,6,7,8. There are several important differences between the fibril and non-fibrous forms of type I collagen, including matrix stiffness and efficiencies of reconstruction of ECM components by the cells during the culture1. Matrix stiffness is one of the most studied regulatory factors of cell culture1, 9. However, the complex interactions between substrates and cells remain to be clarified. To examine the complex interactions between cells and environmental factors, a simple system is useful. Comparison of the cellular behavior on the two different forms of collagen may help to simplify the effect of environmental factors. Depending on the purpose of their use, different forms of type I collagen can be selectively used. Normally, keratinocytes are in contact with the basement membrane but not with type I collagen. However, during wound healing, keratinocytes move to the dermal connective tissue, proliferate, and heal the wound10.
Recently, we demonstrated that the concentration of extracellular calcium is important for proliferation of keratinocyte line cells by using the culture system on the fibril form of type I collagen mimicking the dermal connective tissue11. When the keratinocyte cell line FEPE1L-8 was cultured on the fibril form of type I collagen, the shape of the cells was round and their proliferations were stopped at an extracellular calcium concentration of 30 &#956;M11. When the calcium concentration was increased to 1.8 mM, cell growth was recovered11. The cells grew under both calcium concentrations (30 &#956;M and 1.8 mM) when cultured on the non-fibrous form11, whereas they were more sensitive to the exogenous calcium concentration when cultured on the fibril form. FEPE1L-8 was generated through transfection with the papillomavirus type 16 transforming genes E6 and E7 from human cervical carcinoma, non-tumorigenic, inhibit unlimited proliferation with limited differentiation potential like normal keratinocytes12,13. FEPE1L-8 cells can be maintained by using some kinds of keratinocyte specific medium, including K110 Type-II with additive supplement K-1 (K110)6. Here, we describe the culture protocol of the human keratinocyte cell line on the non-fibrous and fibril forms of type I collagen.

<table border="0" cellpadding="0" cellspacing="0" style="width:1096px;" width="1094">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Albumin, from bovine serum (BSA)</td>
			<td style="width:253px;">Merck KGaA</td>
			<td style="width:127px;">A4503</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">1 % BSA&#160;</td>
			<td style="width:253px;">Merck KGaA</td>
			<td style="width:127px;">A4503</td>
			<td>Dissolve 1 g of BSA powder in 100 mL of PBS (-) and filtrate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Counting Kit-8</td>
			<td>Dojindo Molecular Technologies, Inc.</td>
			<td>347-07621</td>
			<td>tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H&#160;tetrazolium, monosodium salt (WST-8)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen type I (Acid soluble collgen)</td>
			<td>Nippi Inc.</td>
			<td>ASC-1-100-20</td>
			<td>from bovine (any other species available, concentration is 3.0 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">disposable membrane filter unit DISMIC cellulose acetate</td>
			<td>ADVANTEC Co., LTD.</td>
			<td>25CS020AS</td>
			<td>0.2 &#181;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human keratinocyte line cell&#160; FEPE1L-8</td>
			<td></td>
			<td></td>
			<td>&#160;Donated Dr.W.G. Carter (Fred Hutchinson Cancer Research Center, Seattle, WA)&#160;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">K-1</td>
			<td style="width:253px;">Kyokuto Seiyaku Inc.</td>
			<td style="width:127px;">28204</td>
			<td>additive supplement with 500 mg of BSA, 500 mg of bovine pituitarybody extracts, 2.5 mg of insulin, 0.05 &#181;g of h-EGF and 5 mg of heparin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K110 Type-II medium</td>
			<td>Kyokuto Seiyaku Inc.</td>
			<td>28204</td>
			<td>keratinocyte basal culture medium&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">K110 Type-II medium with K-1 and Penicillin-Streptomycin (K110)</td>
			<td style="width:253px;">Kyokuto Seiyaku Inc.</td>
			<td style="width:127px;">28204</td>
			<td>Add 5 mL of Penicillin-Streptomycin and 10 mL of additive supplement (K-1) in 500 mL of K110 type-II keratinocyte basal culture medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">PBS (-)&#160;</td>
			<td style="width:253px;">Merck KGaA</td>
			<td style="width:127px;">P-5368</td>
			<td>Dissolve 1 pouch of PBS (-) powder in 1000 mL of deionized water and filtrate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">10x PBS (-)</td>
			<td style="width:253px;">Merck KGaA</td>
			<td style="width:127px;">P-5368</td>
			<td>Dissolve 1 pouch of PBS (-) powder in 100 mL of deionized water and filtrate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>MP Biomedicals, LLC</td>
			<td>1670049</td>
			<td>10000 units/mL of penisillin G and 10,000&#956;g/mL of streptmycin sulfate in physiological saline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Phosphate bufferred saline BioPerformance CertiCertified, pH 7.4</td>
			<td style="width:253px;">Merck KGaA</td>
			<td style="width:127px;">P-5368-10 pack</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin from porcine pancreas</td>
			<td>Merck KGaA</td>
			<td>T4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">0.05 % trypsin&#160;</td>
			<td style="width:253px;">Merck KGaA</td>
			<td style="width:127px;">T4799</td>
			<td>Dissolve 0.25 g of&#160; trypsin and&#160; 0.186 g of EDTA.2Na in 500 mL of PBS (-)&#160; and filtrate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor from soybean</td>
			<td>FUJIFILM Wako Pure Chemical corporation</td>
			<td>202-20123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor&#160;</td>
			<td>FUJIFILM Wako Pure Chemical corporation</td>
			<td>202-20123</td>
			<td>Dissolve 50 mg of trypsin inhibitor and 500 mg of BSA in 500 mL of PBS (-) and filtrate&#160;</td>
		</tr>
	</tbody>
</table>

evaluation of keratinocyte proliferation on two- and three-dimensional type i collagen substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

A schematic representation of the treatment of the surfaces of culture dishes using type I collagen is depicted in Figure 1. Cell morphologies observed on the non-fibrous and fibril forms are presented in the left- and right-side panels of Figure 2, respectively. FEPE1L-8 cells were cultured for 2 h (upper panels) and 3 days (lower panels). In the initial 2 h of culturing, the cells adhered and spread on both forms of collagen (Figure 2, upper panels). Three days after seeding, cells on the non-fibrous form continued to spread and cell numbers increased (Figure 2, lower left panel). In contrast, the cells on the fibril form showed limited spreading (Figure 2, lower right panel). FEPE1L-8 cells continued to proliferate on the non-fibrous form of type I collagen (Figure 3, solid black line, closed black circles) and on the untreated dish surfaces (Figure 3, dotted gray line, closed gray circles). In contrast, cells did not proliferate on the fibril form (Figure 3, dotted line, open circles). Figure 2 and Figure 3 have been modified from Fujisaki et al11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59339/59339fig1v2.jpg" /> 
Figure 1: Schematic representations of culture dish surfaces treated with type I collagen. Under acidic conditions, type I collagen molecules are adsorbed on the surface of a dish in the non-fibrous form (left panel). Under neutral conditions at 37 &#176;C, type I collagen molecules are reassembled into fibrils and adsorbed on the surface of dishes in the gel form (right panel). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59339/59339fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59339/59339fig2.jpg" /> 
Figure 2: Morphology of FEPE1L-8 cells. FEPE1L-8 cells in K110 were cultured using the non-fibrous form (10 &#181;g/mL; left panels) or fibril form (1 mg/mL; right panels) of type I collagen for 2 h (upper panels) or 3 days (lower panels). White bars indicate 100 &#181;m. Figure 2 has been modified from Fujisaki et al. in Figure 1E&#8211;H11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59339/59339fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59339/59339fig3v3.jpg" /> 
Figure 3: Proliferation of FEPE1L-8 cell. The number of viable cells were estimated on the non-fibrous form (black solid line, black filled circles) or on the fibril form (dotted line, open circle) of type I collagen, or untreated dish surfaces (gray dotted line, gray filled circles) for 2 h, 1 day, and 3 days. Experiments were performed in triplicates and values are shown as means + SD. This figure has been modified from Fujisaki et al. in Figure 1J11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59339/59339fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates at JoVE.com

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

evaluation-keratinocyte-proliferation-on-two-three-dimensional-type-i

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.0K Views.  Nippi Research Institute of Biomatrix. Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Video: Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Summary

Explore More Videos

Chapters in this video

Three-dimensional Optical-resolution Photoacoustic Microscopy

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Planar and Three-Dimensional Printing of Conductive Inks

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates

An Improved Method for the Preparation of Type I Collagen From Skin

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Summary

Explore More Videos

Chapters in this video

Three-dimensional Optical-resolution Photoacoustic Microscopy

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Planar and Three-Dimensional Printing of Conductive Inks

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

An Improved Method for the Preparation of Type I Collagen From Skin

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates