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 etha...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium hydroxide (NH4OH)</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&#39;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 ...

This protocol make it possible to see different types of cultures and observe their interactions with extra-cellular matrix. The main advantage of this method is that the matrix you obtain is regenerated by fibroblast and it will be quite similar to the ones generated in vivo. We believe that this sickness come irrelevant when using fibroblast obtaining from baking dishes.Establishing a matrix from these cells could enlighten us how it can effect, for instance, persistent situation. These methods can also be applied to, for example, physical studies to survivors from matrix proprieties. Visualization of this protocol it's important to observe a pipetting technique of the steps that you have to regent.When trying these methods for the first time it will probably take more time than expected. Our advice is to prepare as much as you can in advance and using duration periods for getting ready for next steps. Begin by preparing 0.2%gelatin, 1%glutaraldehyde and one molar ethanolamine solutions according to the manuscript directions.Making sure to run each solution through a 0.22 micrometer filter prior to use. Next, prepare ascorbic acid and lysis buffer. Dilute the PBS pen-strep as described in the manuscript and prepare DMEM with 10%FBS.Prepare heat-denatured 2%BSA by adding 2 grams of BSA to 100 milliliters of sterile water and warming it in a boiling water bath for seven minutes. Then, supplement FBS with penicillin, streptomycin, gentamicin, and amphotericin B.Culture recombinant telomeres transfected immortalized human foreskin fibroblasts and DMEM with 10%FBS and maintained them at 37 degrees Celsius and 5%carbon dioxide. To establish the primary fibroblast culture, cut the tissue samples into small pieces of approximately two to three milliliters cubed and seed them in FPS with a high concentration of antibiotics.When the first fibroblasts appear, replace the medium with FBM medium for cell maintenance. Add two milliliters of 0.2%gelatin solution to each well of a six-well plate and incubate it for one hour for 37 degrees Celsius or overnight at four degrees Celsius. After the incubation, aspirate the gelatin and wash each well with two milliliters of PBS.Add two milliliters of 1%of glutaraldehyde to each well and incubate the plate at room temperature for thirty minutes which will cross-link the gelatin. Aspirate the glutaraldehyde and wash the wells in two milliliters of PBS for five minutes. To block the remaining glutaraldehyde, add two milliliters of one molar ethanolamine and incubate the plate at room temperature for thirty minutes.Aspirate the ethanolamine and repeat the washes with PBS. Add one milliliter of DMEM with 10%FPS. If the medium immediately turns pink, remove it, wash the wells once with two milliliters of PBS and add another milliliter of DMEM.Seed one milliliter of fibroblast suspension with 5 x 10 to the fifth cells in each well and culture the cells until 100%confluence is reached. Then remove the medium and replace it with DMEM with 10%FBS and 50 micrograms per milliliter ascorbic acid. Replace the medium every two days for six days.Two days after the last ascorbic acid treatment remove the medium and wash the wells with two milliliters of PBS. After the washes, slowly add one milliliter of preheated lysis buffer and incubate the plate at room temperature for five to ten minutes until the fibroblasts are lysed. Without removing the lysis buffer, carefully add two milliliters of PBS to each well and then aspirate approximately 2.5 milliliters.Repeat this process twice for a total of three washes. After the final wash, add two milliliters of PBS with pen-strep. Seal the plate with film and keep it at four degrees Celsius for up to three months.Grow HUVEC cells and EMB-2 with 2%FBS until maximum confluence. Then replace the medium with EBM-2 without FBS for eight hours. To prepare the matrices for cell seeding, remove them from the refrigerator and leave them at room temperature for one hour.Block the matrices by add two milliliters of heat-denatured 2%BSA and incubate them at 37 degrees Celsius for one hour. Then, aspirate the BSA and wash them with two milliliters of PBS. Seed 2 x 10 to the fifth HUVEC cells to each well of the plate coated with fibroblast-derived matrices.Incubate the cells at 37 degrees Celsius for 16 hours then examine the tube-like structures under a standard bright field microscope at 20-40x magnification. This protocol was used to examine the affects of PDGF treatment on fibroblast-derived matrix formation. The BGH third fibroblasts that were incubated with PDGF showed a more aligned cell distribution in matrices than those incubated without PDGF.Collagen 1 and fibronectin protein expression was increased in matrices derived from PDGF-stimulated fibroblasts and consequently matrix thickness was increased. Moreover, directionality histograms demonstrate that collagen 1 and fibronectin show parallel patterns. When HUVEC cells were seeded on to decellularized matrices derived from PDGF-stimulated fibroblasts they developed more capillary-like structures than under none stimulated conditions.Confirming that matrices derived from PDGF-BB stimulated fibroblasts promote endothelial cells activation. When performing this procedure, it's important to prepare solutions in advance and make sure the cells are ready before starting. Besides endothelial cells studies matrices can also be seeded with epithelial cells and the reaction to external factors like condition in media or cytotoxic drugs can be observed.Generation of extra-cellular matrix is crucial in the design of tumor macroenvironment experiments.

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

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7.3K 次观看.  Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), CIBERONC. 该协议可以查看不同类型的区域性并观察它们与细胞外矩阵的相互作用。此方法的主要优点是，您获得的基质由成纤维细胞再生，并且与体内生成的基质非常相似。我们认为，这种疾病在使用从烘焙菜肴获得的成纤维细胞时是无关紧要的。从这些细胞建立一个矩阵可以启发我们如何能影响，例如，持续的情况。这些方法也可以应用于，例如，从矩阵礼节对幸存者进行物?...