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.
