Xtopore

Advanced Method for Three-Dimensional Cell Culture

Xtopore is an advanced method for three-dimensional cell culture. This new culture technology is replacing conventional monolayer cell culture methods.

• Macroporous microcarriers
• Porous chitosan bead
• Non-toxic
• Applicable to high density cell culture
• Applicable to most adherent cells, including NIH3T3, CHO-K1, PC12, Caco-2, COS-1, PA317, primary hepatocyte
• Save the cost of expensive cell culture

Specifications of Xtopore

Particle Diameter: 700-1000 μm
Volume (ml/g dry): 50ml
Aprox. no. of microcarriers (/g dry): 30,000/g dry
Average pore diameter (μm): 50-80 μm

Operating Protocol

• Preparation of chitosan bead (χtopore)
  1. Hydrate and swell the dry χtopore in PBS (pH 7-7.5)
  2. Autoclave the χtopore in PBS at 121°C for 15min.
  3. After autoclaving, allow the sterilized χtopore to settle, remove the supernatant.
  4. Wash with fresh PBS by inverting several times (twice)
  5. Remove the supernatant, add new culture medium containing serum.
  6. Leave them overnight at 4°C (optional).
• Culture procedure
  1. Seed 5X105 - 1X106 cells per 100mm culture plate
  2. Incubate cells 5% CO2, 37°C incubator until 70-80% of plate is covered with cells.
  3. Wash the cell plate with warm PBS once.
  4. Add 1ml of warm trypsin-EDTA solution and incubate 1-3 minutes at 37°C until cells get round.
  5. Add 3ml of warm culture medium and detach cells from the plate by mild pipetting of medium.
  6. Put the cell suspension into 15ml and centrifuge at 1,500 rpm for 2 minutes.
  7. Aspirate the supernatant.
  8. Resuspend cells with appropriate culture medium at the desired cell numbers per unit volume.
  9. Mix the cell suspension with χtopore prepared ready to use. The concentration of χtopore for
     35mm bacteriological petri dish or 15ml centrifugal tube is approximately 30-40 beads/ml and
     the χtopore are usually inoculated with 1X107 cells/3ml.
  10. Rock the χtopore with cell suspension at 37°C under the 5% CO2 for 4-6 hrs during initial culture
      period.
  11. Remove the supernatant with unattached cells.
  12. Add new medium and return the culture plate or tube into the CO2 incubator.
  13. Analyze cell state using classical techniques such as LDH assay, SEM imaging, etc.

Note: χtopore culture conditions should be adjusted with cell types and culture purpose.