Transformed cells, such as tumor cells, have the characteristic feature of anchorage- independent growth, unlike normal cells. Some normal cells, such as chondrocytes, are also capable of anchorage-independent growth, and the phenotypic expression of these cells is known to be stronger compared with monolayer cultures. Soft agar culture is a method in which cultures are grown with cells suspended in soft agar gel, and has been used conventionally as a method to detect the ability of cells to undergo anchorage-independent growth. As agar solidifies on cooling, the temperature must be maintained at approx. 37°C while preparing the seed culture plate. Also, since special reagents are required when harvesting the cells in the gel, the resulting culture is not suitable for analysis of cell function. In contrast, alginate, which is an anionic polysaccharide derived from cell walls of brown algae, form a gel in the presence of calcium and liquefy to a solution upon addition of a calcium chelating agent. Alginate gel has been a choice for three-dimensional (3D) cell culture because only cultured cells can be easily harvested. The Alginate 3D Cell Culture Kit is a convenient, easy-to-manufacture kit optimized to produce alginate gel beads. This product has been used to develop successful 3D cell culture systems for a range of different cells types including tumor cells and chondrocytes.
Easy to produce alginate gel beads and harvest the cultured cells from gel.
Suitable for 3D culture including tumor cells and chondrocytes.
|Sodium alginate solution||25 mL ×1 bottle, sterile|
|Calcium chloride solution||100 mL ×2 bottles, sterile|
|Sodium citrate solution||100 mL ×1 bottle, sterile|
|Plastic flexible needle||4 pieces, sterile|
|24-well plate||4 pieces, for suspension culture, sterile|
Materials and Instruments Required, but Not Included:
1. Physiological saline (sterile)
2. 22G Hypodermic needle [e.g., Terumo, product code (NN-2238R)]
3. 5 mL Syringe [e.g., Terumo, product code (SS-05SZ)]
4. Magnetic stirrer and magnetic stir bar (sterile)*
5. Specimen cup or 100 mL beaker (sterile)*
6. Spatula (sterile) [e.g., Coning, product code (3003)]
*Not required if alginate beads are directly prepared in a 24-well plate
Preparation of Alginate Beads
(1) Prepare cell suspended alginate solution in 5mL syringe.
(2) Drop the cell suspension into each well containing calcium chloride solution and produce alginate beads.
(3) Wash alginate beads.
(4) Dispense culture medium and let start the culture.
Cell Recovery from Alginate Beads
(1) Remove the medium from the well.
(2) Add sodium citrate solution and dissolve the alginate beads.
(3) Centrifuge and harvest the cells as a precipitated pellet.
Normal porcine chondrocytes prepared from articular cartilage of knee joint were cultured in alginate beads at 2x106 cells/mL (in DMEM/F-12 containing 10%FBS, 100ng/mL IGF-I and 25μg/mL L-ascorbic acid) in the presence or absence of oversulfated chondroitin sulfate. At end of culture, the alginate beads were dissolved with sodium citrate solution and the cell pellet was digested with Pronase solution (1mg/mL). The DNA and collagen contents of the digested sample were determined using the Hoechst 33258 fluorescent dye method and measurement of hydroxyproline content, respectively (Tissue Eng Part A. 2010 May; 16:1575-84).
|Porcine Chondrocytes cultured in alginate beads|
2. HepG2 cells
A human liver carcinoma cell line, HepG2 cells, was cultured in alginate beads (5×105 cells/mL, 10 beads/well, DMEM containing 10%FBS) for 9 days.
|HepG2 cell cultured in alginate beads
HepG2 cells were cultured in alginate beads (1×105 cells/mL, 10beads/well, DMEM containing 10%FBS). At end of the culture, the alginate beads were dissolved with sodium citrate solution and the cell pellet was digested with Pronase solution (1mg/mL). The DNA content of the digested sample was determined using the Hoechst 33258 fluorescent dye method.
HepG2 cells were suspended in alginate beads and cultured (2×105cells/mL, 10 beads/well, DMEM containing 10%FBS). After 24 hours, antitumor drug, 5-flurouacil (5-FU), was added and further cultured for 6 days. At end of culture, the alginate beads were dissolved with sodium citrate solution and the cell pellet was digested with Pronase solution (1mg/mL). The DNA content of the digested sample was determined using the Hoechst 33258 fluorescent dye method.
Alginate 3D Cell Culture Kit
Sodium alginate solution
Calcium chloride solution
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.