Name: three-dimensional alginate-bead culture of human pituitary adenoma cells
Uploaded: 2016-02-18T15:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells at JoVE.com

We thank Mr. O. Rios for his technical assistance.

This study was approved for the use of human material by the local ethical committees of the Medical Institution and the Center of Research and Advance Studies of the National Polytechnic Institute.
1. Tumor Sample Acquisition
<ol>
	<li>Obtain the pituitary adenoma tissue biopsy from a patient through a trans-sphenoidal surgical procedure14.</li>
	<li>Collect the pituitary adenoma sample and place it into a sterile glass bottle containing 50 ml of fresh 199 culture medium (M199) e...

<ol>
	<li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+Extracellular+Matrix+Mimics+for+3D+Cell+Culture.">Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture.</a> Biotechnol. Bioeng. 103 (4), 655-663 (2009).</li><li>Amsden, B., Turner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+calcium+alginate+gels.">Diffusion characteristics of calcium alginate gels.</a> Biotechnol. Bioeng. 65 (5), 605-610 (1999).</li><li>Andersen, T., Strand, B., Formo, K., Alsberg, E., Christensen, B., Rauter, A. P., Lindhorst, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginates+as+biomaterials+in+tissue+engineering.">Alginates as biomaterials in tissue engineering.</a> Carbohydrate chemistry. Vol. 37, 227-258 (2012).</li><li>Jonitz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+capacity+of+human+chondrocytes+embedded+in+alginate+matrix.">Differentiation capacity of human chondrocytes embedded in alginate matrix.</a> Connect. Tissue. Res. 52 (6), 503-551 (2011).</li><li>Hill, E., Boontheekul, T., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+scaffolds+to+enhance+transplanted+myoblast+survival+and+migration.">Designing scaffolds to enhance transplanted myoblast survival and migration.</a> Tissue. Eng. 12 (5), 1295-1304 (2006).</li><li>Purcell, E. K., Singh, A., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+composition+effects+on+a+neural+stem+cell-seeded+scaffold.">Alginate composition effects on a neural stem cell-seeded scaffold.</a> Tissue. Eng. Part. C. Methods. 15 (4), 541-550 (2009).</li><li>Wee, S., Gombotz, 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=Protein+release+from+alginate+matrices.">Protein release from alginate matrices.</a> Adv. Drug. Deliv. Rev. 31 (4), 267-285 (1998).</li><li>Tashjian, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+strains+of+hormone-producing+pituitary+cells.">Clonal strains of hormone-producing pituitary cells.</a> Methods. Enzymol. 58, 527-535 (1979).</li><li>Fasekas, I., 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=Characterization+of+human+pituitary+adenomas+in+cell+cultures+by+light+and+electron+microscopic+morphology+and+immunolabeling.">Characterization of human pituitary adenomas in cell cultures by light and electron microscopic morphology and immunolabeling.</a> Folia. Histochem. Cytobiol. 43 (2), 81-90 (2005).</li><li>Kohler, P. O., Bridson, W. E., Rayford, P. L., Kohler, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+Production+by+Human+Pituitary+Adenomas+in+Culture.">Hormone Production by Human Pituitary Adenomas in Culture.</a> Metabolism. 18 (9), 782-788 (1969).</li><li>Kletzkky, O. A., Marrs, R. P., Rundall, T. T., Weiss, M. H., Beierle, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer+and+suspension+culture+of+human+prolactin-secreting+pituitary+adenoma.">Monolayer and suspension culture of human prolactin-secreting pituitary adenoma.</a> Am. J. Obstet. Gynecol. 138 (6), 660-664 (1980).</li><li>Melmed, S., Odenheimer, D., Carlson, H. E., Hershman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+functional+human+pituitary+tumor+cell+cultures.">Establishment of functional human pituitary tumor cell cultures.</a> In Vitro. 18 (1), 35-42 (1982).</li><li>Thompson, K. W., Vincent, M. M., Jensen, F. C., Price, R. T., Schapiro, E. <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+hormones+by+human+anterior+pituitary+cells+in+serial+culture.">Production of hormones by human anterior pituitary cells in serial culture.</a> Proc. Soc. Exp. Biol. Med. 102 (2), 403-408 (1959).</li><li>Cappabianca, P., Cavallo, L. M., Solari, D., Stagno, V., Esposito, F., de Angelis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+endonasal+surgery+for+pituitary+adenomas.">Endoscopic endonasal surgery for pituitary adenomas.</a> World neurosurg. 82 (6 Suppl), S3-S11 (2014).</li><li>Aplin, J., Curtis, A. S. G., Lackie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+Matrices+for+Studing+Cell+Adhesion.">Model Matrices for Studing Cell Adhesion.</a> Measuring cell adhesion. , 110-111 (1991).</li><li>Guiraud, J. 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=Human+prolactin-producing+pituitary+adenomas+in+three-dimensional+culture.">Human prolactin-producing pituitary adenomas in three-dimensional culture.</a> In. Vitro. Cell. Dev. Biol. 27 (3), 188-190 (1991).</li><li>Ma, H. L., Hung, S. C., Lin, S. Y., Chen, Y. L., Lo, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenesis+of+human+mesenchymal+stem+cells+encapsulated+in+alginate+beads.">Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads.</a> J. Biomed. Mater. Res. A. 64 (2), 273-281 (2003).</li></ol>

This protocol has several critical steps. The first is bead size homogeneity, which is required to maintain the same conditions of diffusion of the nutrients and gases in all of the cell culture. In our experience, the use of a 21 G needle to make the alginate beads allows for the acquisition of an efficient culture of uniform bead size. The second important factor is the distance from the needle; this distance must be no more than 5 cm from the calcium solution to avoid deformed pearls and dead cells due to the collisio...

Three-dimensional scaffolds have been extensively used in cell culturing because they provide a three-dimensional structural support for cultured cells and the maintenance of cell-to-cell communication1. Naturally derived polymer materials have been used to make three-dimensional scaffolds including type I collagen, chitosan and alginate. Alginate is a natural polymer derived from the brown sea algae Macrocystispyrifera (Kelp). This polymer is composed of repeated units of &#946;-D-mannuronic acid (M) and &#945;-L-guluronic acid (G)2 and forms stable gels in the presence of certain divalent cations, such as calcium and barium. Calcium chloride (CaCl2) solutions are commonly used as the crosslinking reagent to form alginate beads. Alginate solutions dropped into CaCl2 immediately form three-dimensional spherical gels. When the alginate solution is mixed with cells, the cells are encapsulated into the alginate beads3.
Alginate has properties that have enabled it to be used as a matrix for the encapsulation of a variety of cells, including chondrocytes4, skeletal myoblasts5 and neural stem cells6. It is an inert material and permits the diffusion of nutrients, oxygen and metabolic products that maintain cell survival and function; moreover, unlike other gel-based culture systems, cells cultured within alginate beads can also be liberated from the scaffold using calcium chelators, such as sodium citrate, and the cells can then be harvested for further investigations7.
Pituitary adenomas are typically benign tumors with low proliferation rates. Rat pituitary adenoma cell lines have been successfully cultured in a two-dimensional system8. However, this culture of secretory human pituitary cell tumors is not an efficient development; dispersed tumor pituitary cells grow poorly in culture, the cells exhibit limited attachment and spreading, and the cells typically form floating aggregates in the culture dish9,10.
Several attempts have been made to obtain a tumor pituitary cell suspension, including an enzymatic and mechanical dispersion approach11, a solely mechanical dispersion approach12 and the culturing of tumor explants10. With these approaches, different authors have obtained viable adherent cultures for different periods of time. The capacities of the tumor secretory cells to survive in these two-dimensional systems depend on the tumor type and their proliferation rates9. However, in long-term cultures, cells with fibroblast phenotypes predominate11,13. This paper describes a method for obtaining a primary culture from pituitary adenoma cells encapsulated in alginate beads that further liberates them from the alginate scaffold that enables detailed analyses of aspects of their cell biology, e.g., their cytoskeletal arrangements.

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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.

This protocol has been applied successfully in the culture and maintenance of adenoma pituitary cells in alginate beads for different periods of time. Figure 1 shows embedded cells in alginate beads after three months; these cells exhibit bi-refractive rounded shapes under an inverted light microscope (Figure 1). Figure 2 shows the localization of N-cadherin in rat pituitary adenoma cells embedded in alginate. The N-cadherin is localized ...

Watch this Scientific Journal Video about Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells at JoVE.com

Three-dimensional Alginate-bead Culture of Human 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 ...

The overall goal of this method, is to describe a three-dimensional alginate-bead culture for maintaining human pituitary adenoma cells. This method can help answer key questions in the tumor cell biology field, such as the expression of different markers of invasiveness. The main advantage of this technique is that alginate scaffold, in contrast with other three-dimensional sytems, permits pituitary adenoma cells to ameliorate from the alginate beads for further investigation.Demonstrating the procedure will be Carmen Solano-Agama, a research assistant from my laboratory. To begin, while working inside a laminar flow cabinet, place previously-obtained adenoma tumor tissue in a 35 millimeter dish, containing approximately 20 milliliters of calcium-and magnesium-free PBS. Using surgical tweezers, transfer the tissue into another petri dish containing fresh PBS to wash the tissue.Repeat the transfer into fresh buffer until the red blood cells and debris are removed. Next, with the surgical tweezers, hold the pituitary adenoma tissue, and with a small surgical scissor, cut the tissue into small pieces. Then, using a pasteur pipette with rounded edges, transfer the tissue fragments and buffer into a 15 milliliter tube.Centrifuge at 68 times g at room temperature for 10 minutes. After the spin, use a pasteur pipette to remove the PBS, and add three to five milliliters of collagenase solution. Mix two to three times by inverting the tube, and incubate the tissue in the collagenase at 37 degrees Celcius under constant rotation for 30 minutes.Centrifuge the tube again for 10 minutes, and use a pasteur pipette to remove the collagenase solution. Add 5 milliliters of TNase, and mix two to three times by inverting, then centrifuge the sample again. If the tissue is not completely dissociated, perform a second enzymatic digestion according to the text protocol.Next, aspirate the supernatant and discard it. Then use one volume of EGTA solution to re-suspend the pellet, and gently mix with two volumes of alginate solution. Remove the plunger from a sterile syringe containing a 21 gauge needle, and use a pipette to load the alginate and cell colution into the syringe.Gently insert the plunger into the syringe, and place the syringe needle in the beaker, approximately five centimeters from the calcium chloride solution. Now, carefully dispense the alginate cell suspension drop-by-drop into the beaker. The suspension will jell on contact with the solution to form spherical beads.Using circular motions, slowly stir the glass beaker to prevent the beads from sticking. Then keep the alginate beads in the calcium chloride solution for five minutes. After the incubation, carefully remove the calcium solution, and use three to five milliliters of M199 enriched with 20%of FBS to wash the beads twice.With a sterile spatula, transfer the alginate beads into a regular T25 tissue culture flask, and add four to five milliliters of M199 with FBS. Incubate at 37 degrees Celcius with 5%carbon dioxide in a humidified incubator. After preparing poly-D-lycine coated coverslips, according to the text protocol.To analyze the actin cytoskeleton, use the pipette to transfer one milliliter of alginate beads from the culture flask into a 15 milliliter tube. Add five milliliters of 55 millimolar sodium citrate, and centrifuge at 68 times g at room temperature for 10 minutes. Then aspirate the supernatant, and discard.Using one milliliter of warm M199 enriched with 20%FBS, we suspend the pellet and gently mix the cell suspension. Then, after counting the cells according to the text protocol, seed 35, 000 cells on 13 milliliter diameter poly-D-lycine coated glass coverslips. After incubating the cells for 48 hours, remove the culture medium, and use PBS to wash the cells once.Then, add 500 microliters per coverslip of fixation buffer to fix and permeabilize the cells, and incubate at 37 degrees Celcius for 10 minutes. Remove the fixation buffer and add 500 microliters per coverslip of 3.5%paraformaldehyde in PBS. Then incubate at room temperature for 30 minutes before removing the solution.Finally, after washing the cells and incubating in rhodamine conjugated phalloidin and dapi solution, according to the text protocol, use a confocal laser scanning microscope with a 63x subjective and a 541 excitation wavelength to acquire images of the actin cytoskeleton. This figure shows embedded adenoma pituitary cells in alginate beads after three months. As shown here, the cells exhibit birefractive rounded shapes under an inverted light microscope.In this figure, the localization of N-cadherin in rat pituitary adenoma cells embedded in alginate can be seen. The N-cadherin is localized at cell-to-cell contacts and the nucleus displays the chromatin extended. In this experiment, the proliferation index was obtained from 10 nonfunctioning human pituitary adenomas, using the immune reactivity for Ki67, and a mean labeling index of 19.2 plus or minus 1.5%was obtained.Here, the actin cytoskeleton of noninvasive cells exhibited elongated shapes with small actin stress fibers. The more follages and actin filament arrangements varied with the pituitary adenoma independent of the culture system. For example, in a nonfunctioning invasive adenoma, the predominant shape among these cells was rounded, with discontinuous cortical rings of actin filaments.Once mastered, the primary pituitary adenoma culture can be done in three hours. An alginate encapsulation can be done in 15 minutes if it is performed properly. While attempting this procedure, it's important to remember to maintain the needle no more than five centimeters from the calcium solution to avoid the firm pearls and dead cells due to the collision of the stream of the alginate cell solution with the surface of the calcium solution.Following this procedure, other methods, like fixation of the alginate bits, followed by dehydration with polyethylene glycol can be performed in order to answer additional questions, like how pituitary adenoma cells organize their cytoskeleton in this three-dimensional scaffold. After watching this video, you should have a good understanding of how to maintain pituitary adenoma cells in a three-dimensional alginate bead culture.

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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 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 ...

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

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.

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 ...

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 ...

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 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 ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...