Name: a three-dimensional tissue culture model to study primary human bone marrow and its malignancies
Uploaded: 2014-03-08T15:00:00
Description: Watch this Scientific Journal Video about A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies at JoVE.com

This work was funded by grants from the National Institutes of Health, National Cancer Institute (1R21CA141039), BD Biosciences Research Grant Award, the American Cancer Society Institutional Research Grant (IRG #58-006-53), to the Purdue University Center for Cancer Research, and the Canadian Institutes of Health Research and the Alberta Cancer Board Research Initiatives Program. M.P. was supported by the National Institutes of Health, National Cancer Institute, Cancer Prevention Internship Program (R25CA128770; PI: D. Teegarden) administered by the Oncological Sciences Center and the Discovery Learning Research Center at Purdue University.

1. Advance Preparation
<ol>
	<li>Keep sterile serological pipettes and pipette tips at 4 &#176;C. 
	Troubleshooting: Matrigel gels at room temperature (RT); using cold pipettes and tips will prevent Matrigel from gelling during the culture setup.</li>
	<li>Thaw a bottle of Matrigel at 4 &#176;C overnight.</li>
</ol>
2. Preparation of Reagents
<ol>
	<li>Preparation of fibronectin stock solution: Make a 1 mg/ml stock solution of fibronectin by dissolving 100 mg human fibron...

<ol>
	<li>Carrel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Permanent+Life+of+Tissues+Outside+of+the+Organism.">On the Permanent Life of Tissues Outside of the Organism.</a> J. Exp. Med. 15, 516-528 (1912).</li><li>Harrison, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+living+developing+nerve+fiber.">Observations on the living developing nerve fiber.</a> Proc. Soc. Exp. Biol. Med. 4, 140-143 (1907).</li><li>Earle, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+malignancy+in+vitro.+IV.+The+mouse+fibroblast+cultures+and+changes+seen+in+the+living+cells.">Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells.</a> J. Natl. Cancer Inst. 4, 165-212 (1943).</li><li>Gey, G. O., Coffman, W. D., Kubicek, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Culture+Studies+of+the+Proliferative+Capacity+of+Cervical+Carcinoma+and+Normal+Epithelium.">Tissue Culture Studies of the Proliferative Capacity of Cervical Carcinoma and Normal Epithelium.</a> Cancer Res. 12, 264-265 (1952).</li><li>Barcelloshoff, M. H., Aggeler, J., Ram, T. G., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional-Differentiation+and+Alveolar+Morphogenesis+of+Primary+Mammary+Cultures+on+Reconstituted+Basement-Membrane.">Functional-Differentiation and Alveolar Morphogenesis of Primary Mammary Cultures on Reconstituted Basement-Membrane.</a> Development. 105, 223-227 (1989).</li><li>Lamb, R., Ambler, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocytes+propagated+in+serum-free,+feeder-free+culture+conditions+fail+to+form+stratified+epidermis+in+a+reconstituted+skin+model.">Keratinocytes propagated in serum-free, feeder-free culture conditions fail to form stratified epidermis in a reconstituted skin model.</a> PloS One. 8, (2013).</li><li>Duong, H. S., Le, A. D., Zhang, Q. Z., Messadi, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3-dimensional+culture+system+as+an+in+vitro+model+for+studying+oral+cancer+cell+invasion.">A novel 3-dimensional culture system as an in vitro model for studying oral cancer cell invasion.</a> Int. J. Exp. Pathol. 86, 365-374 (2005).</li><li>Kirshner, 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=A+unique+three-dimensional+model+for+evaluating+the+impact+of+therapy+on+multiple+myeloma.">A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.</a> Blood. 112, 2935-2945 (2008).</li><li>Gudjonsson, T., 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=Normal+and+tumor-derived+myoepithelial+cells+differ+in+their+ability+to+interact+with+luminal+breast+epithelial+cells+for+polarity+and+basement+membrane+deposition.">Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition.</a> J. Cell Sci. 115, 39-50 (2002).</li><li>Kirshner, J., Chen, C. J., Liu, P., Huang, J., Shively, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CEACAM1-4S,+a+cell-cell+adhesion+molecule,+mediates+apoptosis+and+reverts+mammary+carcinoma+cells+to+a+normal+morphogenic+phenotype+in+a+3D+culture.">CEACAM1-4S, a cell-cell adhesion molecule, mediates apoptosis and reverts mammary carcinoma cells to a normal morphogenic phenotype in a 3D culture.</a> Proc. Natl. Acad. Sci U.S.A. 100, 521-526 (2003).</li><li>Weaver, V. 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=Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies.">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> J. Cell Biol. 137, 231-245 (1997).</li><li>Gunn, E. 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=The+natural+products+parthenolide+and+andrographolide+exhibit+anti-cancer+stem+cell+activity+in+multiple+myeloma.">The natural products parthenolide and andrographolide exhibit anti-cancer stem cell activity in multiple myeloma.</a> Leukemia Lymphoma. 52, 1085-1097 (2011).</li><li>Hsiao, A. Y., 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=384+hanging+drop+arrays+give+excellent+Z-factors+and+allow+versatile+formation+of+co-culture+spheroids.">384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids.</a> Biotechnol. Bioeng. 109, 1293-1304 (2012).</li><li>Meads, M. B., Gatenby, R. A., Dalton, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment-mediated+drug+resistance:+a+major+contributor+to+minimal+residual+disease.">Environment-mediated drug resistance: a major contributor to minimal residual disease.</a> Nat. Rev. Cancer. 9, 665-674 (2009).</li><li>Golde, D. W., Cline, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Human+Bone-Marrow+in+Liquid+Culture.">Growth of Human Bone-Marrow in Liquid Culture.</a> Blood. 41, 45-57 (1973).</li><li>Gartner, S., Kaplan, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Culture+of+Human-Bone+Marrow-Cells.">Long-Term Culture of Human-Bone Marrow-Cells.</a> Proc. Natl. Acad. Sci. Biol. 77, 4756-4759 (1980).</li><li>Caligaris-Cappio, F., 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=Role+of+bone+marrow+stromal+cells+in+the+growth+of+human+multiple+myeloma.">Role of bone marrow stromal cells in the growth of human multiple myeloma.</a> Blood. 77, 2688-2693 (1991).</li><li>Pike, B. L., Robinson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+marrow+colony+growth+in+agar-gel.">Human bone marrow colony growth in agar-gel.</a> J. Cell. Physiol. 76, 77-84 (1970).</li><li>Bug, G., 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=Rho+family+small+GTPases+control+migration+of+hematopoietic+progenitor+cells+into+multicellular+spheroids+of+bone+marrow+stroma+cells.">Rho family small GTPases control migration of hematopoietic progenitor cells into multicellular spheroids of bone marrow stroma cells.</a> J. Leukocyte Biol. 72, 837-845 (2002).</li><li>Provenzano, P. P., 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=Collagen+reorganization+at+the+tumor-stromal+interface+facilitates+local+invasion.">Collagen reorganization at the tumor-stromal interface facilitates local invasion.</a> BMC Med. 4, (2006).</li><li>Tancred, T. M., Belch, A. R., Reiman, T., Pilarski, L. M., Kirshner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Expression+of+Fibronectin+and+Collagens+I+and+IV+in+Multiple+Myeloma+and+Monoclonal+Gammopathy+of+Undetermined+Significance.">Altered Expression of Fibronectin and Collagens I and IV in Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance.</a> J. Histochem. Cytochem. 57, 239-247 (2009).</li></ol>

The rBM model provides a comprehensive system where cellular composition of the BM is maintained ex vivo for up to 30 days. BM cells grown in rBM self-stratify according to their function closely mimicking the BM architecture found in vivo. Moreover, this is the first system that allows for the expansion of the multiple myeloma clone8. The most critical steps for ensuring the robust growth of BM cells and the overall success of the culture are: 1) to obtain a healthy population of the BM cell...

Tissue culture was developed to study cell behavior in a controlled environment to minimize the systemic variability when comparing various processes in intact organisms. This method was first established in the early 19001,2 and refers to a technique where tissue explants were cultured ex vivo in a glass dish. In the mid-1900s the system was adapted to grow dispersed cells rather than fragments of intact tissues, and the terms &#39;tissue culture&#8217; and &#39;cell culture&#8217; became synonymous3. In such conventional cell culture systems cells are grown on the surface of the tissue culture plastic overlaid with growth medium supplemented with various growth factors. Two types of cultures have emerged based on the adhesion capacity of various cells: adherent cell culture, where cells attach and spread on the tissue culture plastic and nonadherent cultures, where cells are propagated in suspension. Since the early days of cell culture, multiple immortal cell lines have been created, such as HeLa4, the first human cancer cell line. These cell lines have the capacity to proliferate indefinitely in cell culture, and most do not require any special treatment to maintain viability.
The reductionist approach of the original cell culture methods was designed to simplify the system as much as possible by including only the bare minimum components required to sustain cell viability and proliferation. However, simplified cell culture approaches fail to support ex vivo most primary human cell types with finite life-span. Therefore, new culture systems are being designed to approximate the tissue microenvironment as closely as possible, thus allowing cell explants to grow under physiological conditions. In contrast to the conventional/reductionist cell culture approaches, 3-dimensional (3-D) culture systems are now becoming a preferred method to efficiently culture various human cell lines and primary cells to subsequently study mechanisms involved in health and disease, in the context of a supportive microenvironment. Such 3-D systems are usually set up using reconstructed matrices and/or medium supplemented with growth factors, in order to recapitulate the tissue microenvironment to study cell types of interest. The first of these 3-dimensional (3-D) culture models was developed to study mammary gland development. To provide the cells with native conditions mammary epithelial cells were embedded in Matrigel, collagen IV and laminin-rich source of extracellular matrix (ECM), and overlaid with growth medium. Under such conditions, the mammary epithelial cells formed clusters resembling the mammary acini, and upon stimulation with lactogenic hormones, these acini secreted casein, and other milk proteins, into the hollow lumena of the acini-like structures. Casein secretion was not observed in standard cultures even after addition of prolactin5, further emphasizing the role of microenvironment in preserving the morphological and phenotypic characteristics of cells. Another demonstration of the loss of normal cellular function when cells are taken out of the context of their physiological microenvironment is a demonstration that without supportive microenvironment, keratinocytes fail to form stratified epidermis6. A number of other 3-D models have been created to allow ex vivo propagation of primary cells7,8 .
The crucial role of microenvironment in cell behavior was elegantly demonstrated in a study where mammary epithelial cells formed &#34;inside-out&#34; acini when cultured in collagen I matrix, compared to the correctly polarized acini that were formed in Matrigel. This loss of proper morphology was reverted when laminin-producing myoepithelial cells were added to the collagen I cultures9. Moreover, correct microenvironment is required for the accurate genotype manifestation. Grown in a dish, MCF7 breast cancer cells transfected with a cell-cell adhesion molecule CEACAM1 behave exactly the same as the untransfected CEACAM negative cells. However, when cultured in Matrigel, in contact with ECM, wildtype MCF7 form tumor-like structures while MCF7 cells transfected with CEACAM1 revert to a normal phenotype and form acini with hollow lumena, as has been established with nonmalignant mammary epithelium10. Similarly, blocking &#946;1-integrin in breast cancer cells does not change their behavior under standard culture conditions, but the same experiment performed in 3-D cultures demonstrates that blocking &#946;1-integrin reverts malignant cells to a normal phenotype11. Therefore, tissue microenvironment is not only required to maintain cell viability, but also to retain proper cell function.
In addition to providing a system where cell behavior can be studied under physiological conditions, 3-D cultures function as robust and reliable medium for preclinical testing of novel therapeutics8,12,13. Culturing cells in 3-D allows screening of investigational compounds under the conditions of environment-mediated drug-resistance14, where the contribution of cell-cell and cell-ECM adhesion could be assessed. Furthermore, off-target toxicity of new compounds can be ascertained by incorporating multiple cellular compartments of various tissues. Such screens can be performed more rapidly and are more cost-effective than the comparable studies in vivo8.
Here we present a setup of a 3-D model of reconstructed bone marrow (rBM) where normal and malignant bone marrow (BM) cells proliferate ex vivo in a system closely mimicking the microenvironment of the human BM. Previous attempts at growing primary human BM cells in 2-D cultures, liquid or adherent cocultures with various components of BM stroma, or 3-D, semi-solid agar cultures have met with limited success due to their inability to supply the cells with the components of tissue microenvironment15-18. In these systems primary BM cells had poor viability and failed to proliferate ex vivo. Another system where human BM progenitor cells are grown in spheroid cocultures with BM stromal cells is a 3-D system where hematopoietic stem cell viability and proliferation was sustained for at least 96 hr19. However, although highly beneficial to the understanding of the hematopoietic progenitor biology, this system does not faithfully recapitulate the microenvironment of the BM owing to the absence of ECM components, therefore, limiting its usefulness. The rBM model described here presents a comprehensive system where both cellular and extracellular compartments of the human BM are reconstructed in vitro. We show that rBM cultures can support ex vivo growth of normal human BM cells, as well as cells isolated from patients with various hematological disorders.

<table border="0" cellpadding="0" cellspacing="0" width="877">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">CellTrace CFSE proliferation kit-for flow cytometry</td>
			<td style="width:239px;">Invitrogen/Life technologies</td>
			<td style="width:108px;">C34554</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Fibronectin from human plasma</td>
			<td>Millipore</td>
			<td>NG1884448</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Ficoll-Paque PLUS</td>
			<td>GE Lifesciences</td>
			<td>17-1440-02</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Matrigel basement membrane matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:328px;">Rat tail collagen type I</td>
			<td>BD Biosciences</td>
			<td>354249</td>
			<td style="width:203px;">Collagen I from Millipore does not polimerize properly using this protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">Vectashield mounting medium for fluorescence (regular set)</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">SigmaFast Red TR/Napthol AS-MX tablets</td>
			<td>Sigma-Aldrich</td>
			<td>F4648</td>
			<td style="width:203px;">For TRAP staining</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">SigmaFast BCIP/NBT tablets</td>
			<td>Sigma-Aldrich</td>
			<td>B5655</td>
			<td style="width:203px;">For alkaline phosphatase staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Zeiss confocal LSM 510 microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">Zeiss 510 software</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td style="width:203px;">Image analysis software for confocal analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Zeiss Axiovert 200M inverted microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">MetaMorph software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td style="width:203px;">Image analysis software for Axiovert 200M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">FACSort flow cytometer&#160;</td>
			<td>BD biosciences</td>
			<td></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">CellQuest Pro software&#160;</td>
			<td>BD biosciences</td>
			<td></td>
			<td style="width:203px;">Software for the analysis of flow cytometry data</td>
		</tr>
	</tbody>
</table>

a three-dimensional tissue culture model to study primary human bone marrow and its malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

Since primary bone marrow cells fail to proliferate under standard tissue culture conditions, the rBM system was created to closely mimic the bone microenvironment, providing a system to culture and expand BM cells ex vivo. The major components of the BM extracellular matrix, fibronectin, collagen I, collagen IV, and laminin21, were incorporated into rBM matrix to provide the structural scaffold to this 3-D culture. Endosteum, the interface between the bone and the BM, rich in collagen I and fibronect...

Watch this Scientific Journal Video about A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies at JoVE.com

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Research

JoVE Journal

Medicine

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...