Catalog No.   B-P-00002-2、B-P-00002-4、B-P-00002-10                

Specification  2ml-kit、4ml-kit 、10ml-kit

Storage       Store at 4℃. Keep for two years.   

Product Description

The 3D Cell Culture Hydrogel Kit is a complete set of reagents designed for the microenvironments for 3D cell culture and related applications. The complete control of biomolecular modifications and gel stiffness of this easy-to-use kit allow a great variety of cell culture applications. The polymers can generate hydrogels at fast gelation rate. It is recommended to read the User Guide carefully before setting up the 3D culture gels.

(The hydrogel material is of plant origin, no animal origin)

Applications

• Spheroid formation assays

• Adaptable to any 3D cell culture-based drug screening platforms.

Sample Types

• Tumor cell lines

Kit Contents

Procedures

Preparation of the reagents

=A Gel: Place A Gel in 37℃ water bath for at least 10 min till completely thawed.

=C Buffer (1X): Freshly dilute 10X C buffer with cold serum-free culture medium (e.g. serum-free DMEM) to 1X C buffer before use. (Do NOT dilute C buffer with PBS)

=D Buffer (1X): Freshly dilute 10X D buffer with cold 1x PBS to 1X D buffer before use.

Preparation of 3D cell culture

All steps are performed in laminar flow and the volume ratio of each component is added as indicated.

1. Place the Thermal Conductive Sheet on a ice pack. The sheet can rapidly and evenly cool down the culture plate.

2. Resuspend 1×105~107 cells in 1ml mixture of growth medium and A Gel at 1:1 ratio. For example, resuspend 1×105 cells in 500 mL growth medium and 500 mL A Gel.

Note: Grow cells in appropriate media and culture conditions. Adherent cells should be cultured at ~80% confluency.

3. Place a culture plate/dish on Thermal Conductive Sheet/ice pack. Add 20-40 mL cell suspension mix from step 2 onto each well. The mixture should form gel after 5 min. Note: Test gel formation by gently touching gel with pipette tip, and gel thread should not be pulled out when retracting tips from gel surface.

4. Once gel has formed, add 1mL of cold 1X C Buffer to cover the dome for 15 min.

5. After 15 min incubation, carefully replace C Buffer with culture medium. Replace culture medium the following day.

6. Incubate cells at 37°C in CO2 incubator for 7 to 14 days and observe spheroid formation with microscope. The Culture medium may be replaced every other day as required for proper growth of cells.

Dissolving 3D Gels for spheroid collection

1. Carefully remove culture medium and wash the gel dome with 1X PBS.

2. Carefully remove 1X PBS and add 1 ml 1X D buffer onto gel for 5 min incubation at room temperature.

3. Gently pipet the solution with 1 mL tip until gel dome is completely dissolved.

4. Transfer the spheroid-contained solution to

1.5 mL tubes. Spin down the spheroids at 1,000 rpm for 10 min. Resuspend spheroids in media for use in assay of interest.

Isolation of individual cells from spheroids

1. To isolate individual cells, isolate spheroids first with the procedure Dissolving 3D Gels for spheroid collection described above.

2. Add trypsin-EDTA to spheroids and incubate the solution at 37°C. Pipet the solution until spheroids dissociate completely.

When spheroids dissociate completely, add 3 volumes of 1X PBS and spin down cells with 10 min centrifugation at 1,000 rpm. Remove the supernatant and collect cells for proceeding assays.