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 fibronectin in 100 ml of distilled water. Once the fibronectin has dissolved, sterile filter, aliquot, and store at 4 &#176;C for up to 6 months. 
	Troubleshooting: If fibronectin doesn&#39;t dissolve quickly incubate the solution for a few minutes in a water bath set at 37 &#176;C.</li>
	<li>Preparation of 10x&#160;PBS: Dissolve 80 g sodium chloride NaCl, 14.4 g Na2HPO4, 2 g KCl and 2.4 g KH2PO4 in 1 L of distilled water. Set the pH to 7.4, sterile filter, and store at 4 &#176;C for up to 6 months.</li>
	<li>Preparation of neutralization buffer20: Make a solution containing 100 mM HEPES in 2x&#160;phosphate buffered saline (PBS) using 1 M HEPES and 10x&#160;PBS stock solutions. Adjust the pH of the solution to 7.0. Store at 4 &#176;C for 2-3 months.</li>
	<li>Preparation of 2 mg/ml solution of rat tail collagen type I: Make a 2 mg/ml collagen I solution in the neutralization buffer (step 2.3). Vortex the solution at low speed and mix gently by pipetting up and down. As this collagen is very viscous, avoid generation of air bubbles while pipetting. It is recommended to prepare collagen I solution fresh every time. Keep the solution on ice until use.</li>
	<li>Preparation of endosteal coating (rEnd): Mix 1 mg/ml fibronectin stock with 2 mg/ml collagen I stock (steps 2.1 and 2.4) to a final concentration of 77 &#956;g/ml and 29 &#956;g/ml respectively, in 1x&#160;sterile PBS. Mix and store at 4 &#176;C for up to 1 month.</li>
	<li>Preparation of reconstructed bone matrix (rBM):
	<ol>
		<li>Precool 1.5 ml microcentrifuge tubes on ice. Keep all matrix solutions on ice at all times.</li>
		<li>Mix 4 parts Matrigel (Matrigel concentration varies from lot-to-lot, but the variations were found to be negligible), 2.5 parts of 1 mg/ml fibronectin and 1 part of 2 mg/ml collagen I on ice by first pipetting Matrigel into the tube using cold pipette tips and then add fibronectin and collagen I. Matrigel will start solidifying at temperatures above 4 &#176;C, so work quickly while pipetting Matrigel. Mix the matrix very gently by pipetting up and down, avoid introducing bubbles. Keep the tubes on ice while mixing the rBM.</li>
	</ol>
	</li>
	<li>Preparation of bone marrow growth medium (BMGM): Make 500 ml of BMGM containing 6.2 x 10-4 M CaCl2, 10-6 M sodium succinate, 10-6 M hydrocortisone, 20% human plasma (normal or malignant collected from healthy donors or patients), and 1% penicillin/streptomycin in RPMI-1640. CaCl2, sodium succinate, and hydrocortisone supplements can be stored at -80 &#176;C for &#62;6 months as 1,000x&#160;stock solutions. When growing cell lines, fetal bovine serum (FBS) can be substituted for human plasma.</li>
	<li>Preparation of 10% neutral buffered formalin (NBF): Add 37% formaldehyde stock solution to 1x&#160;PBS at 10% v/v. Mix well and store at room temperature for 2-3 weeks protected from light.</li>
	<li>Preparation of 1% bovine serum albumin (BSA): Weigh appropriate amount of BSA and dissolve it in 1x&#160;PBS. Store at 4 &#176;C and use within 1 week. BSA solution tends to get contaminated if kept refrigerated for longer periods. Alternatively, BSA solution can be aliquoted and frozen at -20 &#176;C for long-term storage. Avoid freeze-thaw cycles.</li>
	<li>Preparation of cell recovery solution (CRS): Make 5 mM EDTA, 1 mM sodium vanadate (Na3VO4) and 1.5 mM sodium fluoride (NaF) solution in 1x&#160;PBS. Sterile filter and store at 4 &#176;C up to 6 months.</li>
</ol>
3. Embedded 3-D Culture of Nonmalignant and Malignant Human BM Cells
<ol>
	<li>Purify primary BM mononuclear cells by Ficoll-Plaque gradient centrifugation per manufacturer&#39;s instructions. 
	Optional step: cells can be labeled with 0.25 &#956;M carboxyfluorescein diacetate, succinimidyl ester (CFSE) per manufacturer&#39;s instructions to follow cell proliferation throughout the culture period. 
	Troubleshooting: If cells die after CFSE staining, titrate the amount of CFSE as the concentration depends on cell type; 0.25 &#956;M works well for primary BM mononuclear cells.</li>
	<li>Add 65 &#956;l of rEnd solution into each well of a 48-well tissue culture treated plate. Spread the solution evenly to cover the entire surface of the well and incubate for &#62;30 min at RT.</li>
	<li>Prepare the cell suspension at a density of 0.5 x 106 cells in 10 &#956;l of 1x&#160;PBS per well of a 48-well plate. In a 1.5 ml microcentrifuge tube mix the cell suspension with 100 &#956;l of rBM matrix per well. Mix the cells and rBM matrix by gently pipetting up and down; avoid introducing bubbles. Keep the cell/matrix mixture on ice to avoid gelling of the matrix. 
	Note: the rBM system is fully scalable; to set&#160;up the system in a non-48-well format, adjust the amounts of all reagents (matrices and medium) based on the surface area of the plate used. Scale the number of cells to be plated accordingly.</li>
	<li>Aspirate the remaining rEnd and add the cell/matrix mixture into the center of a well. Quickly, spread the cell/matrix mixture to cover the entire surface of the well evenly. Place the plate for 30-60 min in 37 &#176;C, 5% CO2 tissue culture incubator to allow the matrix to solidify. Do not shake or disturb the plate during incubation. 
	Troubleshooting: To prevent uneven distribution of cells in rBM, mix well prior to plating. Mix after every 5th well plated to avoid cells settling to the bottom of the tube. Once plated, cells do not sink to the bottom of the well due to surface tension in the matrix. 
	Troubleshooting: To prevent uneven distribution of cells in rBM, mix well prior to plating. Mix after every 5th well plated to avoid cells settling to the bottom of the tube. Once plated, cells do not sink to the bottom of the well due to surface tension in the matrix.</li>
	<li>Prewarm BMGM in a water bath set to 37 &#176;C.</li>
	<li>After 30 min, check to see that the matrix has set properly; it will form a soft gel like layer that does not move when the plate is tilted. Overlay the cell/matrix layer with 1 ml of warm BMGM. To avoid tearing of the matrix pipette the medium very gently (dispensing drop-by-drop) using a serological pipette. Place the assembled rBM culture into a 37 &#176;C, 5% CO2 tissue culture incubator and culture for the desired time period. 
	Note: cell viability is maintained for at least 30 days without changing medium; if medium change is required only change 50% of medium each time. 
	Critical step: It is crucial that the medium is not cold as cold temperature might disturb the already set matrix. Take care not to squirt the medium on top of the matrix at it might break the soft matrix layer. 
	Troubleshooting: If cell viability in the rBM system is low, cell density may have to be determined experimentally when working with cells other than primary mononuclear cells from BM aspirates. When working with cell lines, decrease the cell number 10-fold, as immortalized cell lines proliferate faster than primary cells.</li>
</ol>
4. &#39;In-matrix&#39; Imaging
<ol>
	<li>Light microscopy
	<ol>
		<li>Image the cultured cells directly in rBM using bright field, differential interference contrast (DIC), confocal or any other imaging techniques that allow long-distance imaging as the matrix is approximately 1 mm thick. Use the Z-stack feature available on many of the microscopes to image the entire culture from top to bottom.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining 
	Tip: For the immunostaining protocol below avoid aspirating staining/washing solutions, instead tilt the plate and remove solutions using a pipette.
	<ol>
		<li>Remove the BMGM by using a pipette and wash 2-3x with 100 &#956;l/wash/well of RT 1x&#160;PBS. Make sure that enough PBS is used to cover the entire surface of the well.</li>
		<li>Fix cells in matrix by adding 100 &#956;l/ well (for a 48-well plate) of 10% NBF for 15 min at RT. Remove NBF and wash cells twice with 100 &#956;l/wash/well of RT 1x&#160;PBS. 
		Note: Avoid using cold NBF as it may liquefy the matrix.</li>
		<li>Remove PBS, add 1% BSA for 1 hr at RT or at 4 &#176;C overnight in order to block nonspecific binding of antibodies.</li>
		<li>Prepare primary antibody solution in 1% BSA at the required dilution. Dilution will vary for each antibody, check with the manufacturer or titrate to determine the optimal antibody concentration. Add 100 &#956;l/well of primary antibody solution and incubate according to the manufacturer&#39;s instructions. 
		Critical step: All incubations involving antibodies conjugated to fluorophores or fluorescent dyes should be performed in the dark.</li>
		<li>Remove primary antibody solution and wash 3x with 1x&#160;PBS for 5 min.</li>
		<li>For indirect immunofluorescence staining, incubate with a secondary antibody conjugated to fluorescent probe for 1 hr at RT. Dilute the antibodies in 1% BSA at concentration suggested by the manufacturer.</li>
		<li>Remove the secondary antibody solution and wash 3x with 1x&#160;PBS for 5 min. 
		Troubleshooting: To avoid high background during imaging, titrate the fluorescently conjugated secondary antibodies and use the lowest concentration that provides adequate signal. If high background is still an issue, increase the number and the duration of wash steps, or use directly conjugated primary antibodies, and skip step 4.2.6.</li>
		<li>To stain the nuclei add 4&#39;,6-diamidino-2-phenylindole (DAPI) nuclear staining solution, at the dilution suggested by the manufacturer, and incubate for 7-10 min&#160;at RT.</li>
		<li>Remove nuclear staining solution and wash 2x with 1x&#160;PBS for 5 min.</li>
		<li>Remove the PBS from the last wash and add a drop of mounting medium (regular set), on the sample to preserve fluorescence. Image using appropriate excitation/emission settings on a fluorescent or confocal microscope. 
		Note: Imaging should be done within 2 days of staining to avoid degradation of matrix and loss of rBM integrity and loss of fluorescent signal. If not imaged immediately, stained samples should be kept at 4 &#176;C, protected from light, until imaging.</li>
	</ol>
	</li>
</ol>
5. Isolation of Cells from rBM
<ol>
	<li>Remove medium gently without disturbing the matrix layer. Do not aspirate.</li>
	<li>Wash cells 2x with sterile 1x&#160;PBS. Do not aspirate.</li>
	<li>Add 0.5 ml of ice-cold CRS to each well and remove the entire matrix layer along with the cells, by vigorously pipetting up and down. Collect the cells, matrix, and CRS in a 1.5 ml microcentrifuge tube. Place on ice.</li>
	<li>Add an additional 0.5 ml CRS to the same well and collect all remaining material. Transfer to the same microcentrifuge tube.</li>
	<li>Vortex the mixture of cells, matrix, and CRS and incubate on ice for 1hr. Vortex cells every 15 min to facilitate release from the matrix.</li>
	<li>Check the mixture after 1 hr, no visible clumps of matrix should be present. 
	Troubleshooting: if clumps of matrix are still visible after 1 hr, transfer cells/matrix/CRS mixture to a bigger tube, add additional 2-5 ml of CRS, and incubate for an additional 30-60 min.</li>
	<li>Centrifuge cells in CRS at 1,000 rpm for 5-10 min at 4 &#176;C. Remove the supernatant and resuspend the cell pellet in 1 ml of cold 1x&#160;PBS.</li>
	<li>Centrifuge at 1,000 rpm for 5-10 min and remove the supernatant. Repeat the PBS wash one more time.</li>
	<li>The second PBS wash completes the recovery of cells from matrix and isolated cells can be used for any downstream applications such as DNA/RNA/protein isolation, flow cytometry, in vivo transplantation, etc.</li>
</ol>

<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 authors declare&#160;no competing interests.

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 ...

a-three-dimensional-tissue-culture-model-to-study-primary-human-bone

Research

JoVE Journal

Medicine

16.5K Views.  Purdue University. 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.

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

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 malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer 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 either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

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 interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

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

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

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

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

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 step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

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 cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

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

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

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 brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

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 often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

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 drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

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