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

Summary

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.

Abstract

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.

Introduction

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 β-D-mannuronic acid (M) and α-L-guluronic acid (G)² and forms stable gels in the presence of certain divalent cations, such as calcium and barium. Calcium ch....

Protocol

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

1. Obtain the pituitary adenoma tissue biopsy from a patient through a trans-sphenoidal surgical procedure¹⁴.
2. 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. Maint....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
199 culture medium	Invitrogen	31100-027	For culture, warm in a 37 °C water bath before use
Fetal bovine serum	PAA	A15-751
Sodium bicarbonate (NaHCO3)	Merck	106329
N-(2-Hydroxyethyl) piperazine-N´-2ethanesulfonic acid (HEPES)	Sigma	H-4034
Penicillin/Streptomycin	PAA	P11-010
PBS (Dulbecco's Phosphate Buffered Saline)	Invitrogen	21600-044
Collagenase type I	Worthington	4176
Trypsin	Invitrogen	27250-018
Ethylenediaminetetraacetic acid (EDTA)	Research Organics	3002E
Sorbean trypsin inhibitor	Invitrogen	17075-029
DNAse	Worthington	2139
Alginic acid, From Macrocystis Pyrifera (Kelp)	Sigma	A-2158
Calcium chloride (CaCl2)	J.T. Baker	1332
Sodium citrate (Na3C6H5O7)	J.T. Baker	3646
Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)	Research Organics	9624P
Polyethylene Glycol 6000 (PEG 6000)	Calbiochem	528877
Ethylene glycol tetraacetic acid (EGTA)	Research Organics	9574E
Magnesium Sulfate (MgSO4)	J.T. Baker	2500
Triton-X 100	Sigma	X100
Formaldehyde	J.T. Baker	2106
Paraformaldehyde	Sigma	P6148
Ammonium chloride (NH4Cl)	J.T. Baker	660
BSA Bovine Serum Albumin IgG-Free	Jackson Immuno Research	001-000-161
Phalloidin–Tetramethylrhodamine B isothiocyanate	Sigma	P1951	Toxic. Use gloves to handle this reagent
4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)	Invitrogen	D1306	Toxic. Use gloves to handle this reagent
Sulfuric acid (H2SO4)	J.T. Baker	9681	Highly corrosive. Use gloves to handle this reagent
Sodium hydroxide (NAOH)	Merck	1.06498	Can cause eye and skin irritation.Use gloves to handle this reagent
(3-Aminopropyl)triethoxysilane (APTS)	Sigma	A-3648
Poly-D-lysine hydrobromide	Sigma	P7280
Trypan blue solution	Sigma	T8154
Name	Company	Catalog Number	Comments
Equipment			Toxic: May cause cancer. Use gloves to handle this reagent
Rotator	Boekel Scientific	Model 230300
Centrifuge	DuPont Corporation	Model sorvall TC6
shaker	Lab-line Instruments	Model 314-820