Cyst Formation of MDCK Cells in TrueGel3D™ Hydrogels

Introduction

Improved cell culture techniques from 2D to 3D have provided models that more closely mimic the native physiological condition (gene expression, cellular functions and cell behavior1). The TrueGel3D™ hydrogel system has a complete set of reagents to design chemically defined biomimetic hydrogel that can recapitulate natural extracellular matrix (ECM) environments and provides valuable insight into cellular differentiation and morphogenesis.   

Madin Darby Canine Kidney (MDCK) cells are epithelial cells derived from kidney tissue of an adult female cocker spaniel. These cells serve as models to study epithelial morphogenesis. The objective of this study is to evaluate TrueGel3DTM hydrogel in growing MDCK cells and form spherical cysts.

Some cells need attachment sites for growing in a chemically defined environment. The RGD peptide, composed of arginine, glycine and aspartic acid have been identified as a mediator of cell-substratum and cell-cell interactions. Integrins on the cell surface recognize RGD motif and support cell adhesion. In this MDCK cell cyst formation study, we also investigate the optimum concentration of RGD peptide to study epithelial morphogenesis. The scrambled RGD peptide, that does not know to mediate cell adhesion, is used as a control.

Methods

TrueGel3D™ hydrogel preparation

All steps are performed in sterile hood and the volume ratio of each component is added as indicated below.
 

Components (TRUE5, TRUERGD, TRUESRGD) Final Concentration Volume (µL)  
Water NA 9.85 Cell Suspension Mix (25.5 µL)
TrueGel3D buffer, 10X concentrated, pH 5.5 NA 2.4
FAST-PVA (30 mmol/L) 3.5 mmol/L 3.5
TrueGel3D™ RGD integrin adhesion peptide/ TrueGel3D™ scramble RGD integrin adhesion peptide (4 mmol/L) 0.5 mmol/L 3.75
MDCK cell suspension (2.5 X 106 cells/mL) 1.5 X 104 cells/mL 6.0
PEG non cell-degradable/ (20 mmol/L) 3 mmol/L 4.5  
Total   30.0  

 

  1. MDCK cell suspension was prepared in Eagle’s Minimum Essential Medium (EMEM) containing 10% FBS.
  2. Water, 10X buffer (pH 5.5) and FAST-PVA were mixed in a reaction tube.
  3. TrueGel3DTM RGD integrin adhesion peptide or TrueGel3DTM scramble RGD integrin adhesion peptide was added to the reaction tube and mixed immediately to ensure homogenous distribution. It was followed by incubation for 5 min to allow attachment of the peptide to the maleimide groups of FAST-PVA polymer.
  4. 4.5 µL of PEG non cell-degradable crosslinker was pipetted and spotted on the surface of a sterile 8 chamber slide with glass bottom chamber slide compatible for inverse microscopy.
  5. The cell suspension was transferred to the reaction tube containing the polymer (FAST-PVA) (cell suspension mix).
  6. 25.5 µL of cell suspension mix was transferred to the culture dish containing the 4.5 µL of crosslinker and mixed quickly followed by a 3 min incubation to allow gel formation.
    Note: Gel formation starts a few seconds after mixing.
  7. Once gel has formed, 350-400 µL of the cell culture medium (EMEM with 10% FBS) was added.
  8. The culture plate was placed in the incubator. The medium was replaced after 1 hour, and after was changed every 2-3 days.
  9. Cells in the hydrogel were treated on the 14th day to proceed to confocal microscopy.

Chemical cell fixation and confocal microscopy

  1. TrueGel3D hydrogels containing cells were fixed using 4% paraformaldehyde in PBS (with Ca++/Mg++ ) for 30-60 min and washed twice (10 min each) in 1XPBS (w/o Ca++/Mg++ ).
  2. Hydrogels were incubated with 0.1% (v/v) Triton® X-100 in PBS (w/o Ca++/Mg++ ) for 10 min and washed twice with PBS (w/o Ca++/Mg++ ).
  3. F-Actin staining: hydrogels were incubated with 1.7 µg/mL phalloidin-TRITC in PBS (w/o Ca++/Mg++ )  for 1 hour in dark and washed three times (5 min each) in PBS (w/o Ca++/Mg++ ).
  4. Nuclei Staining: hydrogels were incubated with 1 µmol/L Syto 24 Green® (Invitrogen) for 20 min at room temperature in the dark.
  5. Hydrogels were washed three times (5 min each) with PBS (w/o Ca++/Mg++ ) and stored in PBS (w/o Ca++/Mg++) at 4 0C before confocal laser scanning microscopy analysis.

Results

Cyst counted from confocal images from day 15 samples

Figure 1. Cyst counted from confocal images from day 15 samples. Cyst formation increases with increasing concentration of TrueGel3DTM RGD integrin adhesion peptide.

  • TrueGel3D™ hydrogel supported the growth of MDCK colonies.
  • There’s an increased number of cyst formation with increasing concentration of TrueGel3D™ RGD integrin adhesion peptide, but not with TrueGel3D scramble RGD integrin adhesion peptide or thioglycerol (used as negative control).
  • Optimum concentration of TrueGel3D™ RGD integrin adhesion peptide for optimum MDCK cyst formation:500 µmol/L

Merged, confocal images of cyst formed with MDCK cells

Figure 2. Merged, confocal images of cyst formed with MDCK cells after 15 days of culture in TrueGel3D™ FAST-PVA hydrogel modified with (A) 500 µmol/L of TrueGel3D™ RGD integrin adhesion peptide, (B) 500 µmol/L of scrambled RGD peptide and (C) 500 µmol/L of thioglycerol. Green: nuclei; Red: actin cytoskeleton. Scale bar: 200 µm; Magnification: 60X

  • Single layer of polarized cells enclosing lumen was observed after 15 days of culture in TrueGel3D™ hydrogels FAST-PVA hydrogel with TrueGel3D RGD integrin adhesion peptide.
  • MDCK cysts grown in hydrogels containing TrueGel3D™ scramble RGD integrin adhesion peptide showed lumen but no polarized cells.
  • MDCK colonies grown in thioglycerol modified hydrogels neither showed lumen nor polarized cells.

Discussion

TrueGel3D™ hydrogel FAST-PVA with PEG nondegradable crosslinker can grow and form cysts for MDCK cell lines and generate in-vitro models to study epithelial morphogenesis and polarity. Cyst morphology of single layer polarized cells enclosing a lumen is comparable to that observed in natural collagen. TrueGel3D™ RGD integrin adhesion peptide specifically mediate cell adherence to promote cyst formation.

Cysts with polarized cells share many characteristics including physiological development of epithelial tissues and confocal microscopy. These shared similarities have proved to be a powerful tool to analyze 3D structures of MDCK colonies. Similar hydrogel systems can be used to study pulmonary alveolar function by growing alveolar-like cysts (ALCs)2 and to investigate the etiological role of extracellular matrix proteins in polycystic kidney diseases3.

Materials

     

References

  1. Baker, BM, Chen, CS. Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. J. Cell Sci. 2012;125:3015–3024.
  2. Yu, W, Fang, X, Ewald, A, Wong, K, Hunt, CA, Werb, Z, Matthay, MA, Mostov, K. Formation of cysts by alveolar type II cells in three-dimensional culture reveals a novel mechanism for epithelial morphogenesis. Mol. Biol Cell. 2007;18(5):1693–1700.
  3. Liu, B, Li, C, Liu, Z, Dai, Z, and Tao, Y. Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease. BMC Nephrol. 2012;13:109.