Three-dimensional alginate spheroid culture system of murine osteosarcoma

Abstract

Abstract. Osteosarcoma (OS) is the most common primary malignant tumor of the bone and often forms pulmonary metastases, which are the most important prognostic factor. For further elucidation of the mechanism underlying the progression and metastasis of human OS, a culture system mimicking the microenvironment of the tumor in vivo is needed. We report a novel three-dimensional (3D) alginate spheroid culture system of murine osteosarcoma. Two different metastatic clones, the parental Dunn and its derivative line LM8, which has a higher metastatic potential to the lungs, were encapsulated in alginate beads to develop the 3D culture system. The beads containing murine OS cells were also transplanted into mice to determine their metastatic potential in vivo. In this culture system, murine OS cells encapsulated in alginate beads were able to grow in a 3D structure with cells detaching from the alginate environment. The number of detaching cells was higher in the LM8 cell line than the Dunn cell line. In the in vivo alginate bead transplantation model, the rate of pulmonary metastasis was higher with LM8 cells compared with that of Dunn cells. The cell characteristics and kinetics in this culture system closely reflect the original malignant potential of the cells in vivo.

Transfection of NF-κB decoy oligodeoxynucleotide suppresses pulmonary metastasis by murine osteosarcoma

“…When 490% confluency was reached at day 2, the cells were digested using 0.05% trypsin and encapsulated in 1.2% low-viscosity alginate (Keltone LV; Kelco, Chicago, IL) in 0.15 M sodium chloride (NaCl) at a concentration of 4.0 Â 10 6 cells per ml. 23 Encapsulation was achieved by gently expressing drops of the cell suspension through a 21 gauge needle from a 10 ml syringe into 102 mM calcium chloride; each drop was instantly transformed into a semisolid microspheric bead. After 10 min of incubation to allow further polymerization, the newly formed beads were washed three times with normal saline, followed by one wash with Dulbecco's modified Eagle medium (DMEM: Gibco BRL, Grand Island, NY) to remove excess calcium chloride.…”

“…23 Mice were divided into four experimental groups with 10 mice in each group. The mice were anesthetized with ketamine (80 mg kg À1 ) and xylazine (7 mg kg À1 ).…”

“…The number of detached cells was greater in the LM8 cell line than in the Dunn cell line, suggesting that the cell kinetics in this culture system reflect the in vivo malignancy potential of the cells. 23 To evaluate the effect of the NF-kB decoy ODN on the metastatic potential in vitro using this culture system, the number of cells detaching from the alginate bead and adhering to the bottom of the culture plate was quantified. On day 1 of culture, detached cells were found only in the control group.…”

“…In a preliminary experiment, 23 beads containing murine OS cells, either the parental Dunn cell line or its derivative cell line LM8, were transplanted into the dorsal skin of mice to determine their metastatic potential in vivo. The rate of pulmonary metastasis was higher for LM8 cells than Dunn cells, suggesting that the metastatic potential of cells in alginate bead culture reflect the original in vivo potential.…”

“…21 Therefore, we hypothesized that transfection with NF-B decoy ODN would suppress the tumor growth and pulmonary metastases of osteosarcoma. Using murine osteosarcoma cells from two metastatic clones, the parental Dunn cell line and its derivative LM8 with greater metastatic potential to the lung, 22 we have recently established a novel alginate-encapsulated tumor spheroid model 23 to mimic the in vivo microenvironment. The purpose of this study was to examine the effects of NF-B decoy ODN on tumor progression and metastasis-related gene expression in vitro, as well as pulmonary metastasis in vivo, using the LM8 cell line in the alginateencapsulated tumor spheroid model.…”