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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9.8K vistas.  Center for Research and Advanced Studies (CINVESTAV-IPN). 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.Demonstr...