We thank Marleen De Meulemeester for demonstrating the assay technique in the video film. B. I. R. is a Postdoctoral Research Fellow of the Research Foundation &#8211; Flanders (FWO &#8211; Vlaanderen). L.M.M. is a recipient of an Emmanuel van der Schueren grant from the Flemish League against Cancer (Vlaamse Liga tegen Kanker).

<img alt="Figure 1" src="/files/ftp_upload/52792/52792fig1.jpg" /> 
Figure 1.&#160;Schematic overview of the different assay steps. <a href="https://www.jove.com/files/ftp_upload/52792/52792fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Preparation of Precultured Heart Fragments (PHFs)
<ol>
	<li>Incubate a fertilized chick egg at 37 &#176;C for 9 days. Complete this incubation by a d...

<ol>
	<li>Mareel, M. M., Bracke, M. E., Van Roy, F. M., de Baetselier, P., JR, B. e. r. t. i. n. o. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+cancer+invasion.">Molecular mechanisms of cancer invasion.</a> Encyclopedia of Cancer. 2, 1072-1083 (1997).</li><li>Mareel, M., Kint, J., Meyvisch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+study+of+the+invasion+of+malignant+C3H+mouse+fibroblasts+into+embryonic+chick+heart+in+vitro.">Methods of study of the invasion of malignant C3H mouse fibroblasts into embryonic chick heart in vitro.</a> Virchows Arch. B. Cell Pathol. Incl. Mol. Pathol. 30 (1), 95-111 (1979).</li><li>Albini, A., Noonan, D. 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+&#8216;chemoinvasion&#8217;+assay,+25+years+and+still+going+strong:+the+use+of+reconstituted+basement+membranes+to+study+cell+invasion+and+angiogenesis.">The &#8216;chemoinvasion&#8217; assay, 25 years and still going strong: the use of reconstituted basement membranes to study cell invasion and angiogenesis.</a> Curr. Opin. Cell Biol. 22 (5), 677-689 (2010).</li><li>De Wever, O., 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=Single+cell+and+spheroid+collagen+type+I+invasion+assay.">Single cell and spheroid collagen type I invasion assay.</a> Methods Mol. Biol. 1070, 12-35 (2014).</li><li>Wolff, E., Schneider, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=La+transplantation+prolong&#233;e+d&#8217;un+sarcome+de+souris+sur+des+organes+embryonnaires+de+poulet+cultiv&#233;s+in+vitro.">La transplantation prolong&#233;e d&#8217;un sarcome de souris sur des organes embryonnaires de poulet cultiv&#233;s in vitro.</a> C.R. Seances Soc. Biol. Fil. 151 (7), 1291-1292 (1957).</li><li>Easty, G. C., Easty, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+organ+culture+system+for+the+examination+of+tumor+invasion.">An organ culture system for the examination of tumor invasion.</a> Nature. 199, 1104-1105 (1963).</li><li>Schleich, A. B., Frick, M., Mayer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+invasive+growth+in+vitro.+Human+decidua+graviditatis+confronted+with+established+human+cell+lines+and+primary+human+explants.">Patterns of invasive growth in vitro. Human decidua graviditatis confronted with established human cell lines and primary human explants.</a> J. Natl. Cancer Inst. 56 (2), 221-237 (1976).</li><li>Sys, G. M. L., 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=The+In+ovo+CAM-assay+as+a+Xenograftodel+for+Sarcoma.">The In ovo CAM-assay as a Xenograftodel for Sarcoma.</a> J. Vis. Exp. (77), e50522 (2013).</li><li>Bracke, M. E., Van Larebeke, N. A., Vyncke, B. M., Mareel, M. 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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=Anti-invasive+activity+of+3,7-dimethoxyflavone+in+vitro.">Anti-invasive activity of 3,7-dimethoxyflavone in vitro.</a> J. Pharma. Sci. 83 (9), 1217-1221 (1994).</li><li>Parmar, V. 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=Anti-invasive+activity+of+alkaloids+and+polyphenolics+in+vitro.">Anti-invasive activity of alkaloids and polyphenolics in vitro.</a> Bioorg. Med. Chem. 5 (8), 1609-1619 (1997).</li><li>Gaillard, P. J., MB, V. i. s. s. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+culture+technique+using+embryologic+watch+glasses.">Organ culture technique using embryologic watch glasses.</a> Methods in Medical Research. 2, 241-24 (1951).</li><li>Romeis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Das+mikrotom.">Das mikrotom.</a> Mikroskopische Technik. 15 Verb. Aufl. Kapitel. 7, 114-120 (1948).</li><li>Van Marck, V., 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=P-cadherin+promotes+cell-cell+adhesion+and+counteracts+invasion+in+human+melanoma.">P-cadherin promotes cell-cell adhesion and counteracts invasion in human melanoma.</a> Cancer Res. 65 (19), 8774-8783 (2005).</li><li>Attia, M. A., Weiss, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+spontaneous+mammary+carcinomas+in+mice.+V.+Acquired+tumor+resistance+and+enhancement+in+strain+A+mice+infected+with+mammary+tumor+virus.">Immunology of spontaneous mammary carcinomas in mice. V. Acquired tumor resistance and enhancement in strain A mice infected with mammary tumor virus.</a> Cancer Res. 26 (8), 1787-1800 (1966).</li><li>Katritzky, A. 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=QSAR+modeling+of+anti-invasive+activity+of+organic+compounds+using+structural+descriptors.">QSAR modeling of anti-invasive activity of organic compounds using structural descriptors.</a> Bioorg. Med. Chem. 14 (20), 6933-6939 (2006).</li><li>McKinnell, R. G., Bruyneel, E. A., Mareel, M. M., Seppanen, E. D., Mekala, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+in+vitro+by+explants+of+Lucke+renal+carcinoma+cocultered+with+normal+tissue+is+temperature+dependent.">Invasion in vitro by explants of Lucke renal carcinoma cocultered with normal tissue is temperature dependent.</a> Clin. Exp. Metastasis. 4 (4), 237-243 (1986).</li><li>Wanson, J. C., de Ridder, L., Mosselmans, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+hyperplastic+nodule+cells+from+diethylnitrosamine+treated+cells.">Invasion of hyperplastic nodule cells from diethylnitrosamine treated cells.</a> Cancer Res. 41 (12 Pt 1), 5162-5175 (1981).</li><li>Mareel, M. 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=Effect+of+temperature+on+invasion+of+MO4+mouse+fibrosarcoma+cells+in+organ+culture.">Effect of temperature on invasion of MO4 mouse fibrosarcoma cells in organ culture.</a> Clin. Exp. Metastasis. 2 (2), 107-125 (1984).</li><li>Laerum, O. D., Steinsvag, S., Bjerkvig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+tissue+culture+of+the+central+nervous+system:+recent+developments+and+current+applications.">Cell and tissue culture of the central nervous system: recent developments and current applications.</a> Acta Neurol. Scand. 72 (6), 529-549 (1985).</li><li>Schroyens, W., Mareel, M. M., Dragonetti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+invasiveness+of+human+bladder+cancer+from+cell+lines+and+biopsy+specimens.">In vitro invasiveness of human bladder cancer from cell lines and biopsy specimens.</a> Clin. Exp. Metastasis. 1 (2), 153-162 (1983).</li><li>Mareel, M. M., De Bruyne, G. K., Vandesande, F., Dragonetti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+of+embryonic+chick+heart+invaded+by+malignant+cells+in+three+dimensional+culture.">Immunohistochemical study of embryonic chick heart invaded by malignant cells in three dimensional culture.</a> Invasion Metastasis. 1 (3), 195-204 (1981).</li><li>Bjerkvig, R., Laerum, O. D., Mella, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioma+cell+interactions+with+fetal+rat+brain+aggregates+in+vitro+and+with+brain+tissue+in+vivo.">Glioma cell interactions with fetal rat brain aggregates in vitro and with brain tissue in vivo.</a> Cancer Res. 46 (8), 4071-4079 (1986).</li><li>Bracke, M. E., 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=Confrontation+of+an+invasive+(MO4)+and+a+non-invasive+(MDCK)+cell+line+with+embryonic+chick+heart+fragments+in+serum-free+culture+media.">Confrontation of an invasive (MO4) and a non-invasive (MDCK) cell line with embryonic chick heart fragments in serum-free culture media.</a> In Vitro Cell Dev. Biol. 22 (9), 508-514 (1986).</li><li>Mareel, M. M., Bracke, M. E., Storme, G. A., Grundmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+tumor+spread:+a+brief+overview.">Mechanisms of tumor spread: a brief overview.</a> Cancer Campaign. 9: The Cancer Patient. , 59-64 (1985).</li><li>De Neve, W. J., Storme, G. A., De Bruyne, G. K., Mareel, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+image+analysis+system+for+the+quantification+of+invasion+in+vitro.">An image analysis system for the quantification of invasion in vitro.</a> Clin. Exp. Metast. 3 (2), 87-101 (1985).</li><li>Smolle, J., 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=Quantitative+evaluation+of+melanoma+cell+invasion+in+three-dimensional+confrontation+cultures+in+vitro+using+automated+image+analysis.">Quantitative evaluation of melanoma cell invasion in three-dimensional confrontation cultures in vitro using automated image analysis.</a> J. Invest. Dermatol. 94 (1), 114-119 (1990).</li><li>Bracke, M. E., 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=flavonoid+effect+on+tumor+invasion+and+metastasis.">flavonoid effect on tumor invasion and metastasis.</a> Food Technol. 48 (11), 121-124 (1994).</li><li>Bracke, M. 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The authors declare that they have no competing financial interests.

During the preparation of PHFs, the fragments may not stay in suspension but adhere to the vessel wall; this can be overcome by increasing the volume of the culture medium. If the number of PHFs is too low and their size is too big, decrease the volume of the culture medium. Failure of the test cells to aggregate may be due to fluctuations in the temperature or to microbial infection. Alternatively, an inability to aggregate may be an intrinsic characteristic of the cells. During attachment of the aggregates to PHF, poor...

Invasion is the hallmark of malignant tumors. This activity not only leads to the destruction of surrounding tissues, but is also implicated in metastasis formation. Since cancer patients die from invasion and metastasis, and efficient anti-invasive treatments are still scarce, laboratory assays that mimic the invasion of tumor cells have been developed. The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and allows to study the effects of test compounds on tumor invasion.
The rationale behind the use of the assay is the actual concept that tumors are ecosystems where the neoplastic cells continuously interact with their stroma (host cells and extracellular matrix), and that through these molecular interactions invasion is fine-tuned1. So, in the assay tumor cells are confronted with living embryonic chick heart fragments2, which serve not only as substrates for invasion by the tumor cells, but also as a source of different types of stromal cells and matrix elements. The chick heart contains myocytes, fibroblasts and endothelial cells, and the extracellular matrix is composed of laminin, fibronectin and different types of collagen. In this way, the three-dimensional organ culture technique covers many cellular and molecular interactions implicated in invasion of patient tumors.
The main advantage of the chick heart assay is the implementation of stromal effects. This aspect is more complete than in other invasion assays in vitro that are based on tumor cell invasion into non-living gels composed of basement membrane3 or interstitial matrix4 molecules. The concept of confrontation between tumor cells and normal living host tissue as found in organ culture experimentation has been introduced by several authors including Wolff and Schneider in France5, Easty and Easty in the United Kingdom6, and Schleich in Germany7. Two technical advantages of the chick heart invasion assay over the cited methods is that the volume of the fragments can easily be standardized, and that they remain contractile, which allows functional integrity monitoring during organ culture. Furthermore, avian embryos are preferred, because they can easily be dissected from the sterile content of the egg. The assay has conceptual resemblance to the chick chorioallantois membrane assay8 by offering a complex stromal surrounding to the tumor cells.
The assay has successfully been applied to distinguish between invasive and non-invasive cell variants from the same human tumors such as in the MCF-7 (mammary)9 and HCT-8 (colonic)10 cell line families. The technique is useful to test potentially anti-invasive compounds as well11,12. As further explained, it can be used for the establishment of structure-activity relations of small organic molecules. The assay does, however, not take into account the contribution of immunological cells to cancer invasion. It should be stressed that the technique cannot be considered as a high-throughput analysis system, because of the high number of manipulations, the limited numbers of assay runs (maximum 30 cultures) and the long turn-around time (about 1 month).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ringer&#39;s salt solution</td>
			<td>Braun</td>
			<td>CEO123</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-agar</td>
			<td>Becton Dickinson</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-(hydroxymethyl)-aminomethaan Analar</td>
			<td>VWR</td>
			<td>103157P</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA : albumin from bovine serum, cohn V fract.</td>
			<td>Sigma</td>
			<td>A4503-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-Rega 3</td>
			<td>Life Technologies&#160;</td>
			<td>19993013</td>
			<td></td>
		</tr>
		<tr>
			<td>Gyrotory shakers</td>
			<td>New Brunswick Scientific</td>
			<td>G10 and G33</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 50 ml</td>
			<td>Novolab</td>
			<td>92717</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dishes</td>
			<td>Novolab</td>
			<td>68516</td>
			<td></td>
		</tr>
		<tr>
			<td>Iridectomy scissors</td>
			<td>Rumex</td>
			<td>11-0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Macroscope with calibrated ocular grid</td>
			<td>Wild</td>
			<td>157702</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>International Medical products</td>
			<td>8599956</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>230251-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris&#39; hematoxylin</td>
			<td>Sigma</td>
			<td>HHS32-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslipping resin (Tissue-Tek)</td>
			<td>Sakura</td>
			<td>4494</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin melting apparatus</td>
			<td>GFL</td>
			<td>1052</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome for paraffin sectioning</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stppers for Erlemeyer flasks with gas in- and outlets</td>
			<td>Novolab</td>
			<td>2602421 and 260243</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine</td>
			<td>Sigma</td>
			<td>D8001</td>
			<td></td>
		</tr>
		<tr>
			<td>REAX rocking table</td>
			<td>Heidolph</td>
			<td>54131</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue dishes</td>
			<td>VWR</td>
			<td>NUNC142475</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophtalmological enucleation spoon</td>
			<td>Rumex</td>
			<td>16-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp forceps</td>
			<td>Rumex</td>
			<td>4-111T</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>Nickel-Electro LTD</td>
			<td>7112</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 5 ml</td>
			<td>Novolab</td>
			<td>211901</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides washed and degreased</td>
			<td>International Medical products</td>
			<td>8613908</td>
			<td></td>
		</tr>
	</tbody>
</table>

chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

&#160; The histological sections as presented in Figure 5 show the end result of a number of successful assays. The sound histology of the cultures indicates viable cells and allows to interpret the interaction between tumor cells and normal tissue. Furthermore, no immune reaction from the normal tissue can be observed, which confirms the correct age of the chick embryos used e.g.,&#160;before the immune rejection system is developed. There are no bacteria visible, which indicates absence of (gross) contaminatio...

Watch this Scientific Journal Video about Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds at JoVE.com

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

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8.9K visualizações.  University of Ghent.  Aqui, apresentamos um protocolo para estudar a invasão das células tumorais em fragmentos de tecido normal vivendo em três dimensões. Esta técnica de cultura de órgãos é aplicada principalmente para testar drogas potencialmente anti-invasivos in vitro. 