Name: three-dimensional alginate-bead culture of human pituitary adenoma cells
Uploaded: 2016-02-18T15:00:00.000+00:00
Duration: 8 min 31 s
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) enriched with 10% fetal bovine serum (FBS), 0.6 mg/ml sodium bicarbonate, 2.4 mg/ml HEPES and 1% antibiotics (penicillin/streptomycin) at pH 7.4. Maintain the sample in a glass bottle on ice. 
	Note: The tumor tissue can be processed several hr after the surgery.</li>
	<li>Transport the tumor sample to the laboratory in the glass bottle on ice.</li>
</ol>
2. Preparation of the Enzymatic Tissue Dissociation Solutions, the Trypsin Inhibitor Solution and the Alginate Solutions
Note: Prior to the cell culture procedure prepare the following solutions. Filter all the solutions using a 0.22 &#181;m membrane filter, before use.
<ol>
	<li>Prepare collagenase 1% solution by dissolving 5 mg of collagenase (type I) in 5 ml of M199-serum free.</li>
	<li>Prepare trypsin 0.01% solution by dissolving 0.01 g of trypsin in 10 ml of PBS-EDTA 0.3%.</li>
	<li>Prepare 0.1% trypsin inhibitor solution by dissolving 2 mg of soy trypsin inhibitor in 20 ml of M199-serum free.</li>
	<li>Prepare DNAse solution by dissolving 20 &#181;l of DNAse I 134 U/ml in 4 ml of the trypsin inhibitor solution.</li>
	<li>Prepare EGTA solution (1.2 M) by dissolving 22.8 mg of EGTA in 40 ml of 155 mM NaCl. Adjust the pH to 7.4. Continue to a final volume of 50 ml.</li>
	<li>Prepare HEPES solution (20 mM) by dissolving 238 mg HEPES in 50 ml of 155 mM NaCl.</li>
	<li>Prepare 1.2% alginate solution by dissolving 1.2 g sodium alginate in 100 ml of the HEPES solution. Autoclave the alginate solution.</li>
	<li>Prepare calcium solution (102 mM) by dissolving 750 mg of CaCl2 in 50 ml HEPES solution, adjust pH to 7.4</li>
</ol>
3. Primary Cell Culture and Alginate Encapsulation
<ol>
	<li>Inside a laminar flow cabinet, place the tumor tissue in a 35 mm Petri dish containing approximately 20 ml of PBS Ca2+ and Mg2+ free, pH 7.4 (PBS*).</li>
	<li>Take the tissue with a surgical tweezer and place it in another petri dish containing fresh PBS*, gently wash the tissue. Repeat this step until the red blood cells and debris are removed.</li>
	<li>Using a surgical tweezer, hold the pituitary adenoma tissue, and with a small surgical scissor, cut it into small pieces. With a Pasteur pipette with rounded edges, transfer the tissue fragments and the PBS* into a 15 ml tube, Centrifuge at 68 x g for 10 min at RT. 
	Note: To round the edges of a Pasteur pipette, slightly flame the tip of the pipette in a Bunsen burner.</li>
	<li>Remove the PBS* using a Pasteur pipette and add 3-5 ml of the collagenase solution (approximately 5 ml for 65 mm3 of tissue), mix two to three times by inverting. Incubate the tissue in the collagenase for 30 min at 37 &#176;C in constant rotation.</li>
	<li>Centrifuge the tube at 68 x g for 10 min at RT and remove the collagenase with a Pasteur pipette.</li>
	<li>Add 5 ml of DNAse and mix two to three times by inverting, then centrifuge the tube at 68 x g for 10 min at RT. 
	Note: If the tissue is not completely dissociated, perform a second enzymatic digestion with trypsin for 3 min at 37 &#176;C. Stop the digestion by the addition of 2 volumes of trypsin inhibitor solution.</li>
	<li>Aspirate the supernatant with a Pasteur pipette and discard. Re-suspend the pellet in 1 volume of EGTA solution and mix with 2 volumes of alginate solution.</li>
	<li>Gently mix the cell suspension and alginate by pipetting with a Pasteur pipette. Take a sterile syringe with a 21 G needle, remove the plunger; use a pipette to load the syringe with the alginate and cell solution.</li>
	<li>Gently insert the plunger into the syringe and carefully dispense the alginate-cell solution drop-by-drop into a glass beaker containing approximately 30 ml of the calcium-rich solution.</li>
	<li>Place the needle of the syringe approximately 5 cm up to the CaCl2 solution. The alginate-cell suspension gels on contact with the CaCl2 to form spherical beads. Keep the alginate beads in the calcium solution for 5 min. 
	Note: Stir the glass beaker in which the beads are dropped. Move it slowly with the hand in circular motions, to prevent them from sticking to one another.</li>
	<li>Carefully remove the calcium solution with a Pasteur pipette, and wash the beads twice with 3-5 ml of M199 enriched with 20 % of FBS.</li>
	<li>Transfer the alginate beads into a regular T25 tissue culture flask using a sterile spatula, add 4-5 ml of M199 enriched with 20% FBS and incubated at 37 &#176;C in a humidified incubator with 5% CO2. Change the culture medium every third day.</li>
</ol>
4. Liberate Cells from the Alginate
Note: Prior to liberating the cells from the alginate beads, coat 13 mm diameter coverslips with poly-D-lysine.
CAUTION: SULPHURIC ACID IS A HIGHLY CORROSIVE ACID, AND APTS CAN CAUSE EYE AND SKIN IRRITATION. USE LATEX GLOVES TO HANDLE THESE REAGENTS.
<ol>
	<li>In a fume hood, with a point tweezer immerse the coverslips in a glass petri dish containing 20% aqueous sulphuric acid for 1 hr at RT.</li>
	<li>Decant the sulphuric acid, and wash the coverslips five times with distilled water for 5 min each. Using a point tweezer place the clean coverslips in a petri dish containing 0.1 M NaOH for 5 min at RT15.</li>
	<li>Decant the NaOH and wash one time with distilled water, aspirate the water and dry the coverslips.</li>
	<li>Use a micropipette to coat the coverslips with APTS (aminopropyl-triethoxysilane) for 4 min (use 500 &#181;l for 6 coverslips) flood the Petri dish with distilled water. Quickly aspirate the water and continue to wash with distilled water, moving the coverslips with point tweezers to allow the water to penetrate beneath.</li>
	<li>Aspirate the water and dry the coverslips, using a point tweezer place the coverslips into a clean Petri dish and sterilize them inside the microwave during 3 times, 45 sec each. Add 1,000 &#181;l of sterile water to 100 &#181;g of poly-D-lysine (0.1 mg/ml).</li>
	<li>Incubate the coverslips with approximately 50 &#181;l of poly-D-lysine for 5 min at RT. Aspirate the poly-D-lysine completely. 
	Note: This is important because soluble poly-D-Lysine in the culture medium can inhibit cell proliferation.</li>
	<li>Rinse the coverslips five times with sterile water for 5 min each. Aspirate the sterile water and allow coverslips to dry inside a laminar flow cabinet.</li>
	<li>To analyze the actin cytoskeleton, liberate the pituitary cells from the alginate. Take some of the alginate beads from the culture flask using a 1 ml pipette tip and place them into a 15 ml tube.</li>
	<li>Add 5 ml of 55 mM sodium citrate (dissolve 323.6 mg of Na3C6H5O7, 2 H2O in 20 ml HEPES solution) and centrifuge at 68 x g for 10 min at RT. Aspirate the supernatant and discard.</li>
	<li>Re-suspend the pellet in 1 ml of warm M199 enriched with 20% FSB; gently mix the cell suspension with a Pasteur pipette. 
	CAUTION: PHALLOIDIN AND DAPI ARE TOXIC. USE GLOVES TO HANDLE THESE REAGENTS.</li>
	<li>To count the cells, transfer 125 &#181;l of 0.4% Trypan blue solution to a 1.5 ml tube. Add 75 &#181;l of M199 and 50 &#181;l of the cell suspension obtained in the last step (dilution factor = 5). Mix thoroughly.</li>
	<li>Use a micropipette tip to place approximately 10 &#181;l of the Trypan blue-cell suspension in the hemocytometer chamber.</li>
	<li>Count the cells in the hemocytometer counter using an inverted microscope with phase-contrast illumination. Count all the cells in the 1 mm center square and four 1 mm corner squares. Obtain the cell density per milliliter by multiplying the average count per square by the dilution factor - 104. For example if the average counts per square is 45 cells x 5 x 104 = 2.25 x 106 cells/ml. 
	Note: Count the cells in ten squares of the hemocytometer chamber. Do not count cells touching the middle line at bottom and right sides.</li>
	<li>Seed 35,000 cells on 13 mm diameter glass coverslips covered with poly-D-Lysine.</li>
</ol>
5. Actin Cytoskeleton Arrangement
<ol>
	<li>After 48 hr in culture, remove the culture medium with a Pasteur pipette and quickly add 500 &#181;l per coverslip of PBS, immediately remove the PBS.</li>
	<li>Fix and permeabilize the cells with 500 &#181;l per coverslip of fixation buffer (0.1 M PIPES, pH 6.75, 4% PEG-6000, 1 mM EGTA, 1 mM MgSO4, 0.5 % Triton X-100 and 2% formaldehyde) for 10 min at 37 &#176;C</li>
	<li>Remove the fixation buffer and add 500 &#181;l per coverslip of 3.5% paraformaldehyde in PBS for 30 min at RT. Remove the paraformaldehyde solution.</li>
	<li>Wash the coverslips three times by adding 500 &#181;l per coverslip of PBS for 5 min each. Rinse the coverslips at RT, in constant agitation.</li>
	<li>Incubate the cells with 50 mM ammonium chloride for 10 min, aspirate the ammonium chloride solution and repeat step 5.4.</li>
	<li>Incubate the cells with 1% BSA (IgG-free, proteases-free) in PBS for 30 min, remove the BSA solution and repeat step 5.4.</li>
	<li>Incubate the cells with 50 &#181;M of rhodamine-conjugated phalloidin for 7 min at RT, repeat step 5.4.</li>
	<li>Incubate the cells with 255 &#181;M of DAPI diluted in PBS for 5 min at RT, repeat step 5.4. Finally mount the coverslips with a mounting medium and seal them with nail polish.</li>
	<li>Acquire images of the actin cytoskeletal arrangements using a confocal laser scanning microscope with a 63X objective, at 541 nm excitation wavelength.</li>
</ol>

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The authors have nothing to disclose.

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 collision of the stream of the alginate/cell solution with the surface of the calcium solution. A third critical step is the glass stirring in which the beads are dropped to prevent them from sticking to one another. Sticking results in uneven cell encapsulation.
Some modifications to this protocol can be performed depending of the research question, for example, in our experience the solution of sodium alginate can be mixed with a basement membrane protein like type IV collagen, and following the protocol previously described, obtain a three-dimensional alginate scaffold . Another possibility is to make floating alginate cushions in 15 mm diameter wells; inside these gels, cells can be seeded above or inside them. Troubleshooting when working with the alginate could be necessary if any particular experiment needs modifications in their Ca2+ or Mg2+ concentrations. This is because the hydrogel stability can be altered. Ba2+-and Cu2+-crosslinked alginate gels are relatively stable in aqueous solutions, unfortunately, these cations are often cytotoxic3.
When working with tumor tissues such as pituitary adenoma tissues, one limitation of this technique is the small tissue sample provided. Therefore, the number of alginate beads obtained depends of the size of the tissue sample.
Previously, a system to maintain pituitary adenoma cells in a three-dimensional culture has been described. The authors submitted the cells to gyratory shaking to form aggregates and maintained the cells in this condition for different periods16. The use of this cell suspension culture requires gyratory equipment inside the culture chamber. One advantage of the protocol described here is that it allows for the culturing of cells in a three-dimensional system without the need for extra laboratory equipment. The cells embedded in the three-dimensional alginate system are maintained in a regular incubator inside a regular flask. Another advantage is that the alginate scaffold, in contrast with other three-dimensional systems, permits the cells to be liberated from the hydrogel for further investigation7. Furthermore, an important aspect of culturing cells in alginate beads is that the cells embedded in this system are able to synthetize de novo extracellular matrix. An alternative method for the study of cells embedded in alginate is the fixation of the alginate beads followed by their dehydration with increasing concentrations of polyethylene glycol for subsequent sectioning and Immuno-histochemical analyses17.
We are interested in the future to apply the three-dimensional alginate beads system to embedded primary culture normal pituitary rat cells in order to analyze their interactions in a three-dimensional environment, and also to culture other type of pituitary adenomas.

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.

<table><tbody><tr><td>&lt;strong&gt;Reagent&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>199 culture medium&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>31100-027</td><td>For culture, warm in a 37 &amp;deg;C water bath before use&amp;nbsp;</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>PAA</td><td>A15-751</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck</td><td>106329</td><td></td></tr><tr><td>N-(2-Hydroxyethyl) piperazine-N&amp;acute;-2ethanesulfonic acid (HEPES)</td><td>Sigma&amp;nbsp;</td><td>H-4034</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>PAA</td><td>P11-010</td><td></td></tr><tr><td>PBS (Dulbecco&#039;s Phosphate Buffered Saline)</td><td>Invitrogen&amp;nbsp;</td><td>21600-044</td><td></td></tr><tr><td>Collagenase type I</td><td>Worthington</td><td>4176</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>27250-018</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Research Organics</td><td>3002E</td><td></td></tr><tr><td>Sorbean trypsin inhibitor&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>17075-029</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>2139</td><td></td></tr><tr><td>Alginic acid, From Macrocystis Pyrifera (Kelp)</td><td>Sigma&amp;nbsp;</td><td>A-2158</td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>J.T. Baker</td><td>1332</td><td></td></tr><tr><td>Sodium citrate (Na3C6H5O7)</td><td>J.T. Baker</td><td>3646</td><td></td></tr><tr><td>Piperazine-N,N&amp;prime;-bis(2-ethanesulfonic acid) (PIPES)</td><td>Research Organics</td><td>9624P</td><td></td></tr><tr><td>Polyethylene Glycol 6000 (PEG 6000)</td><td>Calbiochem</td><td>528877</td><td></td></tr><tr><td>Ethylene glycol tetraacetic acid (EGTA)</td><td>Research Organics</td><td>9574E</td><td></td></tr><tr><td>Magnesium Sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>J.T. Baker</td><td>2500</td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>Formaldehyde</td><td>J.T. Baker</td><td>2106</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>J.T. Baker</td><td>660</td><td></td></tr><tr><td>BSA Bovine Serum Albumin IgG-Free</td><td>Jackson Immuno Research</td><td>001-000-161</td><td></td></tr><tr><td>Phalloidin&amp;ndash;Tetramethylrhodamine B isothiocyanate</td><td>Sigma</td><td>P1951</td><td>Toxic. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td><td>Invitrogen&amp;nbsp;</td><td>D1306</td><td>Toxic. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>J.T. Baker</td><td>9681</td><td>Highly corrosive. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>Sodium hydroxide (NAOH)</td><td>Merck</td><td>1.06498</td><td>Can cause eye and skin irritation.Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>(3-Aminopropyl)triethoxysilane (APTS)</td><td>Sigma</td><td>A-3648</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma</td><td>P7280</td><td></td></tr><tr><td>Trypan blue solution</td><td>Sigma</td><td>T8154&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td>Toxic: May cause cancer. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>Rotator&amp;nbsp;</td><td>Boekel Scientific</td><td>Model 230300</td><td></td></tr><tr><td>Centrifuge</td><td>DuPont Corporation&amp;nbsp;</td><td>Model sorvall TC6</td><td></td></tr><tr><td>shaker&amp;nbsp;</td><td>Lab-line Instruments</td><td>Model 314-820&amp;nbsp;</td><td></td></tr></tbody></table>

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 at cell-cell contacts (Figure 2A and 2C) and the nucleus showed the chromatin extended (Figure 2B and 2C).
Tumor cells in alginate beads remain viable for up to 4 months in culture; therefore, the cells can be liberated from the alginate beads at different times, which allows for the analyses of different aspects of the cell biology in the same cell culture. For example, the proliferation index was obtained from ten non-functioning human pituitary adenomas using the immune-reactivity for Ki-67, and a mean labeling index of 19.2 &#177; 1.5% (mean &#177; S.E.M) was obtained. Figure 3 shows cultured human pituitary adenoma cells immunostained for Ki-67.
The actin cytoskeletons of cells of a non-invasive were stained according to the immunocytochemical protocol describe above. Culture cells exhibited elongated shapes with small actin stress fibers (Figure 4A).The morphologies and actin filament arrangements varied with the pituitary adenoma independent of the culture system; for example, in a non-functioning invasive adenoma, the predominant shape among these cells was rounded with an arrangement of their actin filaments in discontinuous cortical rings. (Figure 4B).
<img alt="Figure 1" src="/files/ftp_upload/53637/53637fig1.jpg" /> 
Figure 1. Cells embedded in alginate beads for 3 months. Cells exhibit bi-refractive rounded shapes under an inverted light microscope. In the right panel invasive macroadenoma cells embedded in alginate are shown, and in the left panel non-invasive macroadenoma cells are shown. Scale bar = 15 &#181;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53637/53637fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53637/53637fig2.jpg" /> 
Figure 2. Immuno-cytochemical analysis of N-cadherin in Rat pituitary adenoma cells (GH3) culture in alginate beads. GH3 cells were cultured in alginate, fixed embedded in the alginate system stained for N-cadherin (red) and the nucleus (blue). Cells are arrange in a cumulus showing N-cadherin at the cell-cell borders (A and C). The nucleus (B and C) are shown with the chromatin extended. Scale bar = 15 &#181;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53637/53637fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53637/53637fig3.jpg" /> 
Figure 3. Immunostaining of nuclear antigen ki-67 cultured human pituitary adenoma cells. After 2.5 months in alginate bead culture, invasive non-functioning adenoma cells were fixed and then immunostained for Ki-67 (Red) and the nucleus (blue). Some adenoma cells show positive immunostaining for Ki-67. Scale bar = 15 &#181;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53637/53637fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53637/53637fig4.jpg" /> 
Figure 4. Actin cytoskeleton arrangements of pituitary adenoma cells liberated from alginate beads. Cells were fixed and stained for actin filaments with TRITC-phalloidin. Cells from a non-invasive macroadenoma were extended over the substrate, showing small actin stress fibers (A). Cells from an invasive macroadenoma showed a rounded shape with discontinuous actin rings (B). Scale bar = 15 &#181;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53637/53637fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

three-dimensional-alginate-bead-culture-human-pituitary-adenoma

Nanyang Technological University

Research

JoVE Journal

Medicine

9.9K Views.  Center for Research and Advanced Studies (CINVESTAV-IPN). 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.

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

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Bioengineering

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Cancer Research

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

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 ...

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 ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

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 ...

Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells

Source: Koss, K. M, et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56615">Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation.</a> J. Vis. Exp. (2017).
This video demonstrates a technique to establish a three-dimensional culture of glial cells. Rat-brain-derived glial cells are encapsulated within a photocrosslinkable polymer matrix containing extracellular matrix components. Upon incubating in a growth medium, the glial cells proliferate within the supporting matrix to create a three-dimensional culture.

Stworzenie trójwymiarowej hodowli komórek glejowych pochodzących z mózgu szczura

The protocol for the brain tissue extraction from day 1 Sprague Dawley rat pups, euthanized by decapitation, was approved by the Animal Care and Use ...