The development of this protocol was supported by PI12/02037, PI15/02101, PI17/01847, PI18/01034 and RD12/0036/0041 from the Instituto de Salud Carlos III; by the Fondo Europeo de Desarrollo Regional (FEDER); by &#34;CIBER de C&#225;ncer&#34;, CB16/12/00273 and CB16/12/00446, from the Instituto de Salud Carlos III-FEDER; and by the Fundaci&#243;n Cient&#237;fica AECC (a multifaceted approach to target pancreatic cancer). Cristina Pe&#241;a is a recipient of a Miguel Servet Contract from the Instituto de Salud Carlos III. M. Eaude helped with the English text. We thank lab members for help and advice throughout this research.

Human tissue samples were obtained with the approval of the Research Ethics Board of the Hospital Ram&#243;n y Cajal, Madrid.
1. Preparation of Solutions
<ol>
	<li>Prepare 0.2% gelatin solution: add 1 g of gelatin to 500 mL of PBS. Autoclave the solution and keep at 4 &#176;C. Filter with a 0.22 &#181;m filter before use.</li>
	<li>Prepare 1% glutaraldehyde: Add 1 mL of 25% glutaraldehyde stock solution to 24 mL of PBS. Filter with a 0.22 &#181;m filter before use.</li>
	<li>Prepare 1 M ethanolamine: prepare ethanolamine solution with sterile H2O. Filter with a 0.22 &#181;m filter before use. 
	NOTE: As ethanolamine is provided with a security cap, a needle and syringe will be needed.</li>
	<li>Prepare ascorbic acid: add 0.1 mL of 50 mg/mL stock solution ascorbic acid (light-sensitive) to 100 mL of medium.</li>
	<li>Prepare lysis buffer: prepare PBS 0.5% Triton 100X with 20 nM of NH4OH. Add NH4OH right before use.</li>
	<li>Prepare PBS Pen/Strep: dilute Pen/Strep stock solution to 100 U/mL Pen and 100 &#181;g/mL Strep.</li>
	<li>Prepare DMEM with 10% FBS: Add 10% FBS to 500 mL of DMEM. Supplement with 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 0.1 mg/mL Normocin and 0.25 &#181;g/mL amphotericin B.</li>
	<li>Prepare 2% BSA heat denatured: add 2 g of BSA to 100 mL of sterile water. Warm in boiling water for 7 min.</li>
	<li>Prepare FBS with antibiotics: supplement FBS with 200 U/mL penicillin, 200 &#181;g/mL streptomycin, 100 &#181;g/mL gentamicin and 2.5 g/mL amphotericin B.</li>
</ol>
2. Cell Culture Preparation
<ol>
	<li>Immortalized fibroblasts cell line
	<ol>
		<li>Culture recombinant telomerase transfected immortalized human foreskin fibroblasts (BJ-hTERT, ATCC CRL-4001) in DMEM with 10% FBS and maintain at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Endothelial cells
	<ol>
		<li>Culture human umbilical vein endothelial cells (HUVECs, ATCC PCS-100-013) in EBM-2 medium containing 2% FBS and maintain at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Fibroblast primary culture 
	NOTE: For CAF establishment and culture, the protocol previously published by our group was followed6.
	<ol>
		<li>Briefly, cut tissue samples into small pieces of approximately 2-3 mm3 and seed in FBS with high concentration of antibiotics.</li>
		<li>When the first fibroblasts appeared, replace medium with FBM medium for cell maintenance. 
		NOTE: Depending on tissue origin, cell cultures can be contaminated easily. Wash samples in PBS supplemented with antibiotics, with highly contaminated tissue (e.g., colon6) and shake for 30-45 min.</li>
	</ol>
	</li>
</ol>
3. Fibroblast-derived 3D Matrices (Adapted from Castell&#243;-Cros and Cukierman16)
<ol>
	<li>Add 2 mL of 0.2% gelatin solution to each well of a 6-well plate and incubate for 1 h at 37 &#176;C or overnight at 4 &#176;C.</li>
	<li>Aspirate gelatin and wash it in 2 mL of PBS.</li>
	<li>Add 2 mL of 1% glutaraldehyde and incubate for 30 min at RT. Glutaraldehyde will cross-link gelatin.</li>
	<li>Aspirate glutaraldehyde and wash wells in 2 mL of PBS for 5 min. Repeat 3 times.</li>
	<li>Add 2 mL of 1 M ethanolamine and incubate for 30 min at RT. Ethanolamine will act to block the remaining glutaraldehyde.</li>
	<li>Aspirate ethanolamine and wash wells in 2 mL of PBS for 5 min. Repeat 3 times.</li>
	<li>Add 1 mL of DMEM with 10% FBS. If the medium immediately turns pink, remove the medium, wash in 2 mL of PBS and add again 1 mL of DMEM with 10% FBS.</li>
	<li>Seed 1 mL of fibroblast suspension, with 5 x 105 cells in each well. Total volume in each well will be 2 mL.</li>
	<li>Culture cells until 100% confluence is reached. Then remove the medium and replace with DMEM with 10% FBS with 50 &#181;g/mL ascorbic acid and additional treatment if used.</li>
	<li>Replace medium with fresh DMEM with 10% FBS and 50 &#181;g/mL ascorbic acid every 2 days for 6 days. 
	NOTE: If additional treatment is used (e.g., PDGF-BB, TGF-&#946; and/or other grow factors), add them the same days as ascorbic acid treatment is added.</li>
	<li>Two days after the last ascorbic acid treatment, remove the medium and wash in 2 mL of PBS.</li>
	<li>Slowly add 1 mL of lysis buffer, pre-heated at 37 &#176;C, to each well. Incubate for 5-10 min at RT until fibroblasts are lysed (observable under the microscope).</li>
	<li>Carefully and without removing lysis buffer, add 2 mL of PBS. Then aspirate approximately 2.5 mL of PBS. Repeat twice for a total of three washes.</li>
	<li>Eventually, remove 2.5 mL of PBS and add 2 mL of PBS with Pen/Strep (100 U/mL and 100 &#181;g/mL, respectively). Seal with film and keep at 4 &#176;C for up to 3 months. 
	NOTE: Depending on the experiment, long-term storage of the generated matrices may affect results. We recommend using matrices as soon as possible, and even more so if the experiment involves cell seeding, due to possible protein degradation. If matrices are used for structural assays, such as collagen observation, matrices can be fixed and stored for longer periods. This protocol is indicated for a 6-well culture plate. Other plates can be used, but reactive volumes and cell suspension concentration need to be recalculated according to well area.</li>
</ol>
4. Tube Formation Assay
<ol>
	<li>Grow HUVEC cells in EBM-2 2% FBS until maximum confluence.</li>
	<li>Replace medium with EBM-2 without FBS for 8 h.</li>
	<li>Prepare matrices before seeding cells.
	<ol>
		<li>Remove matrices from the refrigerator and place for 1 h at RT.</li>
		<li>Block matrices by adding 2 mL of heat-denatured 2% BSA. Incubate 1 h at 37 &#176;C.</li>
		<li>Aspirate BSA and wash in 2 mL of PBS.</li>
	</ol>
	</li>
	<li>Seed 2 x 105 HUVEC cells in FBS-depleted medium on each well of a 6-well plate previously coated with 3D fibroblast-derived matrices.</li>
	<li>Incubate endothelial cells at 37 &#176;C for 16 h.</li>
	<li>Examine tube-like structure formation under a standard bright field microscope at 20-40x magnification.</li>
</ol>

<ol>
	<li>Frantz, C., Stewart, K. M., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+at+a+glance.">The extracellular matrix at a glance.</a> Journal of Cell Science. 123 (24), 4195-4200 (2010).</li><li>Kular, J. K., Basu, S., Sharma, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix:+Structure,+composition,+age-related+differences,+tools+for+analysis+and+applications+for+tissue+engineering.">The extracellular matrix: Structure, composition, age-related differences, tools for analysis and applications for tissue engineering.</a> Journal of Tissue Engineering. 5, (2014).</li><li>Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+and+function+of+fibroblasts+in+cancer.">The biology and function of fibroblasts in cancer.</a> Nature Reviews Cancer. 16 (9), 582-598 (2016).</li><li>Erdogan, B., Webb, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts+modulate+growth+factor+signaling+and+extracellular+matrix+remodeling+to+regulate+tumor+metastasis.">Cancer-associated fibroblasts modulate growth factor signaling and extracellular matrix remodeling to regulate tumor metastasis.</a> Biochemical Society Transactions. 45 (1), 229-236 (2017).</li><li>Erez, N., Truitt, M., Olson, P., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Associated+Fibroblasts+Are+Activated+in+Incipient+Neoplasia+to+Orchestrate+Tumor-Promoting+Inflammation+in+an+NF-kB-Dependent+Manner.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kB-Dependent Manner.</a> Cancer Cell. 17 (2), 135-147 (2010).</li><li>Herrera, M., 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=Colon+Cancer-associated+Fibroblast+Establishment+and+Culture+Growth.">Colon Cancer-associated Fibroblast Establishment and Culture Growth.</a> Bio-protocol. 6 (7), e1773 (2016).</li><li>Herrera, A., 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=Endothelial+cell+activation+on+3D-matrices+derived+from+PDGF-BB-stimulated+fibroblasts+is+mediated+by+Snail1.">Endothelial cell activation on 3D-matrices derived from PDGF-BB-stimulated fibroblasts is mediated by Snail1.</a> Oncogenesis. 7 (9), 76 (2018).</li><li>MacColl, E., Khalil, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Metalloproteinases+as+Regulators+of+Vein+Structure+and+Function:+Implications+in+Chronic+Venous+Disease.">Matrix Metalloproteinases as Regulators of Vein Structure and Function: Implications in Chronic Venous Disease.</a> The Journal of Pharmacology and Experimental Therapeutics. 355 (3), 410-428 (2015).</li><li>Shian, S. G., Kao, Y. R., Wu, F. Y. H., Wu, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Invasion+and+Angiogenesis+by+Zinc-Chelating+Agent+Disulfiram.">Inhibition of Invasion and Angiogenesis by Zinc-Chelating Agent Disulfiram.</a> Molecular Pharmacology. 64 (5), 1076-1084 (2003).</li><li>Neri, 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=Cancer+cell+invasion+driven+by+extracellular+matrix+remodeling+is+dependent+on+the+properties+of+cancer-associated+fibroblasts.">Cancer cell invasion driven by extracellular matrix remodeling is dependent on the properties of cancer-associated fibroblasts.</a> Journal of Cancer Research and Clinical Oncology. 142 (2), 437-446 (2016).</li><li>Du, P., Subbiah, R., Park, J. H., Park, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+morphogenesis+of+human+umbilical+vein+endothelial+cells+on+cell-derived+macromolecular+matrix+microenvironment.">Vascular morphogenesis of human umbilical vein endothelial cells on cell-derived macromolecular matrix microenvironment.</a> Tissue Engineering. (Part A), (2014).</li><li>Bignon, M., 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=Lysyl+oxidase-like+protein+2+regulates+sprouting+angiogenesis+and+type+IV+collagen+assembly+in+the+endothelial+basement+membrane.">Lysyl oxidase-like protein 2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane.</a> Blood. 118 (14), 3979-3989 (2011).</li><li>Huang, L., Xu, A. M., Liu, S., Liu, W., Li, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts+in+digestive+tumors.">Cancer-associated fibroblasts in digestive tumors.</a> World Journal of Gastroenterology. 20 (47), 17804-17818 (2014).</li><li>Herrera, A., Herrera, M., Pe&#241;a, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+Snail1+in+the+tumor+stroma.">The emerging role of Snail1 in the tumor stroma.</a> Clinical and Translational Oncology. 18 (9), 872-877 (2016).</li><li>Sheng, Y., Fei, D., Leiiei, G., Xiaosong, G. <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+Scaffolds+for+Tissue+Engineering+and+Regenerative+Medicine.">Extracellular Matrix Scaffolds for Tissue Engineering and Regenerative Medicine.</a> Current Stem Cell Research &amp; Therapy. 12 (3), 233-246 (2017).</li><li>Castell&#243;-Cros, R., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromagenesis+during+tumorigenesis:+characterization+of+tumor-associated+fibroblasts+and+stroma-derived+3D+matrices.">Stromagenesis during tumorigenesis: characterization of tumor-associated fibroblasts and stroma-derived 3D matrices.</a> Methods in Molecular Biology. 522, 275-305 (2009).</li></ol>

The authors declare no conflict of interest.

Matrices can be generated with immortalized fibroblasts or primary fibroblast cultures. Fibroblasts are easy to maintain in in vitro culture with a high growth rate and stress resistance. They can even be isolated from post-mortem tissue3, although contamination could be a restriction, depending on the tissue origin.
The 3D-matrix model here offers a novel and effective system to successfully study ECM composition and fiber orientation. It is also suitable for the analy...

The extracellular matrix (ECM) is a dynamic structure present in all types of tissue. It consists of proteins and polysaccharides that create a net of fibers crucial for cell adhesion, migration, and communication1. ECM composition varies depending on the tissue. While type-I collagen is the most prevalent structural protein, collagen types II, III, V and XI can also be found in various tissues2. Fibronectin generated by fibroblasts is needed for cell adhesion2. Moreover, there are other structural molecules like elastin, laminin and surface receptors called integrins that mediate fiber assembly and are specific to the different ECM tissues2. The ECM plays an important role as a cell scaffold and can also be involved in both physiological and pathological processes1. Abnormalities in the ECM are observed in pathologies such as cancer, which alters ECM composition and/or its organization. In tumors, the ECM represents the non-cellular component of the tumor microenvironment (TME), a complex milieu of cell components such as fibroblasts, immune cells, endothelial cells, pericytes and a variety of soluble factors. It is known that the TME promotes cancer progression and metastasis; cancer-associated fibroblasts, as a predominant cell type in the tumor stroma, take part in this process3. Unlike normal fibroblasts, CAFs are in permanent activation, showing increased secretion of ECM proteins and growth factors (e.g., Transforming Growth Factor-&#946;, TGF-&#946;), as well as a higher expression of some markers, such as &#945;-smooth muscle actin (&#945;-SMA) and fibroblast activation protein (FAP)4. However, CAFs are a heterogeneous cell population, showing different levels of activation or marker expression5. It can be assumed then that the composition and structure of fibroblast-derived matrices will depend on fibroblast status and characteristics.
In this context, the goal of this methodology is to establish an appropriate in vitro model for ECM generation by fibroblasts equivalent to the in vivo ECM setting. We propose this approach as an in vitro translational methodology for further studies of tumor cell functions, like chemoresistance or migration, mediated by ECM. As our group has published elsewhere, CAFs can be obtained from fresh tissue samples, but it has to be noted that the CAFs&#39; survival in culture is limited and their cell passage number is reduced6. In addition, CAF primary cultures established from patients&#39; samples can be used for matrix generation. Manipulation of gene expression in fibroblasts is also an interesting way to produce varied in vitro matrices to assess the possible effects on matrix composition, fiber orientation, etc. Along these lines, our group has recently reported the role of Snail-expressing fibroblasts in the composition and fiber orientation of various derived matrices7.
Furthermore, CAFs and ECM are involved in the vascular system, both in vessel generation and as part of the vase outer layer8. ECM remodeling induces angiogenesis; matrix metalloproteinases (MMPs) seem to be the most important enzyme type contributing to this process9,10. The tissue vascularization of primary cells that generate the ECMs, ECM macromolecules, residual growth factors included in the ECM, matrix elasticity, and matrix thickness are described as factors involved in endothelial cell activation11. In tumors, hypoxia increases ECM stiffness and endothelial sprout generation12. Moreover, CAFs secrete vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) that stimulate angiogenesis in tumor stroma13. In this field, in vitro matrix generation could be used to study angiogenesis processes or MMP action under different experimental conditions. Thus, the in vitro reproduction of the most analogous in vivo matrix could be a valuable tool to investigate ECM&#39;s role in angiogenesis or micro-environmental cell interactions.
The stimulation of cultured fibroblasts with ascorbic acid to enhance matrix deposition and generate an ECM is an accepted way of producing analogous in vivo matrices. Immortalized fibroblast cell lines are easily cultured and are activated by diverse growth factors, like PDGF-BB, Tumor Necrosis Factor-&#945; (TNF-&#945;) or TGF-&#946;14. Within the TME, CAFs synthesize type-I collagen and fibronectin as the main components of ECM4. Similarly, these components are found as major components of in vitro-generated fibroblast-derived matrices (Figure 1).
There are different in vitro methodologies to simulate in vivo ECM. The use of coated culture dishes with mixtures of ECM fibers was extended in past years, but this 2D approach needed improvement to 3D structures, such as cross-linked gels (e.g., Matrigel)1. The Matrigel-like setup has become the standard method for simulating a 3D matrix. Fibrin is also an alternative when generating matrices but fails in terms of strength and durability of the ECM1. Collagen used in combination with other ECM components amends some of the abovementioned issues. However, these collagen gels form a strong network with fibers that can be oriented, but are highly heterogeneous, which can be a problem in experiment repetitions1. Nevertheless, it must be assumed that, depending on the objective of the experiments, the use of Matrigel or other hydrogels is more appropriate (e.g., in matrix contraction studies in which gel contraction can be easily detected).
The potential immunogenicity of the generated matrices could be an issue in experiments with some cell types. Therefore, to reduce the possibility of immune responses due to ECM-generating cells when using our method, matrices are decellularized and washed, although cell fragment removal could not be total15. The ideal ECM needs to be compatible with cell culture and able to communicate and react to cell signals. Our procedure allows the introduction of changes without difficulty during ECM production (e.g., adding fibroblast-stimulating growth factors).

<table><tbody><tr><td>Ammonium hydroxide (NH&lt;sub&gt;4&lt;/sub&gt;OH)</td><td>Roth</td><td>A990.1</td><td></td></tr><tr><td>Amphotericin-B</td><td>Corning</td><td>30-003-CF</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A7906-50G</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Corning</td><td>10-014-CVR</td><td></td></tr><tr><td>Endothelial Basal Medium (EBM)</td><td>Lonza</td><td>CC-3121</td><td>add supplements before use</td></tr><tr><td>Endothelial cell Basal Medium Supplements (EGM-2)</td><td>Lonza</td><td>CC-4176</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma</td><td>411000-100ML</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biowest</td><td>S181B-500</td><td>heat-inactivated before use</td></tr><tr><td>Fibroblast Growth Basal Medium (FBM)</td><td>Lonza</td><td>CC-3131</td><td>add supplements before use</td></tr><tr><td>Fibroblast Growth Medium Supplements and Growth Factors (FGM-2)</td><td>Lonza</td><td>CC-4126</td><td></td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma</td><td>G9391-100G</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>g5882</td><td></td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma</td><td>A92902-100G</td><td>light sensitive</td></tr><tr><td>L-Glutamine</td><td>Lonza</td><td>BE17-605E</td><td></td></tr><tr><td>Normocin</td><td>Invivogen</td><td>3ANT-NR-2</td><td></td></tr><tr><td>Pencillin/Streptomycin (Pen/Strep)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Corning</td><td>21-040-CVR</td><td></td></tr><tr><td>Triton X100</td><td>Roth</td><td>3051</td><td></td></tr><tr><td>Recombinant telomerase transfected immortalized human foreskin fibroblasts (BJ-hTERT)</td><td>ATCC</td><td>ATCC CRL-4001</td><td></td></tr><tr><td>Human umbilical vein endothelial cells (HUVECs)</td><td>ATCC</td><td>PCS-100-013</td><td></td></tr></tbody></table>

fibroblast-derived 3d matrix system applicable to endothelial tube formation assay

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

PDGF-BB stimulated fibroblasts create a thicker ECM. Herrera et al. showed how PDGF-stimulated fibroblasts generated a thicker matrix as well as higher fiber orientation7. BJ-hTERT fibroblasts were incubated with or without PDGF and representative areas observed showed a more aligned cell distribution in matrices produced by PDGF-stimulated fibroblast (Figure 1a). Collagen I and fibronectin protein expression was incre...

Watch this Scientific Journal Video about Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay at JoVE.com

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

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

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

fibroblast-derived-3d-matrix-system-applicable-to-endothelial-tube

Research

JoVE Journal

Cancer Research

7.4K Views.  Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), CIBERONC. Dit protocol maakt het mogelijk om verschillende soorten culturen te zien en hun interacties met extracellulaire matrix te observeren. Het belangrijkste voordeel van deze methode is dat de matrix die u verkrijgt wordt geregenereerd door fibroblast en het zal vrij gelijkaardig zijn aan de matrix die in vivo wordt gegenereerd. Wij zijn van mening dat deze ziekte niet relevant zijn bij het gebruik van fibroblast verkrijgen van het bakken van gerechten.Het vaststellen van een matrix uit de...

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

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Biology

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Bioengineering

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Developmental Biology

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Medicine

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

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

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