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Protocol 1:
MDCK cyst formation in 3-D Life Hydrogels

 1. Introductory Notes


2. Embed cells in hydrogel

Reagents and materials

3-D Life products:

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



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


Experimental procedure:

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-Polymer, 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. 2.4 7.2 = Reagent mix A
Water n.a. 12.8 38.4
Mal-Dextran or Mal-PVA
(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
Cell suspension
(2.5x 106 cells/ml)
1.5x 104 cells 6.0 18.0
(20 mmol/L SH groups)
3 mmol/L
SH groups
4.5 3x 4.5  

Note: The volumes of gel components of any gel composition can be easily determined with an online calculator.


All steps below are performed under a sterile hood:

Instead of the RGD-Peptide, Thioglycerol can be added as described above. In this case the gel does not provide cell attachment sites and can be used as a control to RGD Peptide-modified gels. Cellendes also offers a scrambled version of RGD Peptide for control experiments (3-D Life Scrambled Peptide, Cat # 09-P-003).


3. Fluorescent staining of the actin cytoskeleton and nuclei

Reagents and materials (not included in the 3-D Life product):


Experimental procedure:

Perform all steps that include fixative under a fume hood! In all washing and incubation steps use volumes of solutions large enough to completely cover the hydrogels.


Figure 1: Confocal laser-scanning microscopy of MDCK cell aggregates after 14 days of incubation in PVA-PEG Hydrogel modified with 1 mmol/l RGD Peptide (A) or Thioglycerol (B), respectively. Red: Actin. Green: nuclei.

Note: This result is also obtained by using the Dextran-PEG Hydrogel and 0.5 mmol/L RGD Peptide as described above.