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Protocol 3:
Co-culture of tumor and stroma cells in 3-D Life Hydrogels

1. Introductory Notes


2. Setup of 3-D co-culture

Reagents and materials

3-D Life products:

Reagents and materials not included in the 3-D Life products:



Note: Do not expose 3-D Life thiol-containing reagents (RGD Peptide, CD-Link) to air longer than necessary to avoid oxidation of the thiol-groups. Close cap after each use.


Embed cells in hydrogel:

The volumes of gel components for 30 µL gels are listed in Table 1. If several gels with the same composition are generated, a reagent mix (Reagent Mix A) consisting of 10x CB, Water, Mal-Dextran, RGD Peptide and the cell suspension can be prepared in multiples of the indicated volumes (see 3x example in Table 1).

Note: We recommend preparing the reagent mix with one excess volume of each component to avoid a potential shortage of reagent mix in case slight inaccuracies in pipetting occur.

Table 1: Reagent volumes for 30 µL gels crosslinked with 3 mmol/L maleimide- and SH groups, and modified with 0.5 mmol/L RGD Peptide.

3-D Life reagentsFinal concentrations
in the gel
1x (µL)3x (µL) 
10x CB, pH 5.5 n.a. 2.0 6.0 = Reagent mix A
Water n.a. 9.2 27.6
(30 mmol/l maleimide groups)
3.5 mmol/L
maleimide groups
3.5 10.5
(20 mmol/l SH groups)
0.5 mmol/L
SH groups
0.8 2.4
MCF-7 Cell suspension
(6x 105 cells/ml)
1x 105 cells/mL 5.0 15.0
Fibroblasts cell suspension
(2x 106 cells/ml)
3.3x 105 cells/mL 5.0 15.0
(20 mmol/l SH groups)
3 mmol/L
SH groups
4.5 3x 4.5  

Note: The ratio of tumor cells to fibroblasts in this culture is 1:3.3.


All steps below are performed under a sterile hood: