Hydrogels for 3D cell culture

Introduction

Cells in their natural environment are surrounded by a complex network of extracellular molecules called Extra Cellular Matrix (ECM). This network provides structure and function, in the form of biochemical interactions, to the surrounding cells. Because of limitations raised by classical 2D cell culture technique where this environment is missing (or widely reduced), different approaches have been developed to tempt to mimic this extracellular environment. Among these diverse approaches (scaffold based like hydrogels or rigid scaffolds, scaffold free like low attachment plates or hanging drops for spheroids formation) the most widely used one is hydrogels.

What is a hydrogel?

Hydrogels can be defined as water-swollen networks of polymers. Most are liquids at 4 degrees or room temperature but will form a gel when incubated at 37°C. Because of their properties, cells can be embedded inside hydrogels by mixing cell solution with hydrogel before gel formation: the mix is then dispensed in a cell culture vessel and during the gelation process cells will be encapsulated inside the gel. Compared to classical, 2D cell culture, cells cultured in 3D, embedded in gels, recover different characteristics they have when placed in their natural environment.

comparison between 2D cell culture and 3D cell culture

Figure 1: comparison between 2D cell culture and 3D cell culture. This picture shows the main differences between cells behaviour and constraints when cultivated in 2D environment, plated on a slide, even coated with hydrogels or any other ECM protein like collagen, compared to cells cultivated in a 3D environment, embedded in an hydrogel or any other ECM proteins.

Hydrogels Features

  • Allow the cells to be placed in a more physiological shape (3 dimensions) compared to classical, flat 2 dimension cell culture
  • Some hydrogels can be customized to better mimic the natural environment
  • Stiffness/rigidity of the environment can be adjusted to match with natural stiffness of cell’s tissue of origin
  • No need of sophisticated protocols, material or devices to grow cells in hydrogels

Hydrogel selection guide

The first generation of hydrogels used for 3D cell culture was use of ECM components; it was rapidly followed by more sophisticated systems, generated by doing an extraction of ECM polymers from Engelbreth-Holm-Swarm (EHS) murine sarcoma cell basal membranes (ECM based hydrogels). New generations of synthetic, hybrid or peptides-based materials have been developed since to meet specific requirements and to allow choosing the best option for each cells and applications. However, these hydrogels all show advantages and disadvantages: table 1 summarize the mains characteristics of the different hydrogel platform available at Sigma Aldrich:
 

  ECM based hydrogels (ECM gel, Maxgel…) Synthetic peptide hydrogel (hydromatrix) TrueGel3D biomimetic synthetic hydrogels Natural biomimetic hydrogel: Hystem platform (hyaluronic acid based)
Biological relevance +++ +/- ++ +++
reproducibility +/- +++ +++ ++
Easy to use + +/- ++ +
Potential contaminations ++ - - +/-
Potential Biological reactivity* +++ - - ++
Customization options - +/- +++ +
Cell retrieval +/- ++ +++ +
Downstream analysis (Imaging, molecular analysis) + ++ ++ ++
Data availables (protocols, application notes) +/- +/- +++ ++

*some hydrogels are biologically active and can interact with the cells. It can sometime interfere with the results of your experiments

Table 1: Hydrogels Features

ECM gel (E1270, E6909): Based on the original protocols for ECM extraction from EHS murine sarcoma cell basal membranes, these hydrogel provide a very rich environment, highly compatible with cell development, but they contain mouse growth factors (E6909 is a growth factor reduced version of E1270). Major  components are laminin, type IV collagen, heparan sulfate proteoglycan and entactin. These hydrogels are forming gels by thermic activation between 20 and 40°C and the gelation process is reversible. (protein concentration: 8-12 mg / ml). ECM gel have been successfully used for several applications including analysis of contractility and invasion potential of mammary cancer cell line2, or for In vitro capillary network formation3.

MaxGel Human ECM (E0282): MaxGel human ECM is a human-based alternative to EHS murine sarcoma cell basement membrane extract. It is produced by in vitro co-culture of human fibroblasts and human epithelial cells followed by basement membrane components extraction. Like ECM gels or Cultrex gels, it contains extracellular matrix components including collagens, laminin, fibronectin, tenascin, elastin, a number of proteoglycans and glycosaminoglycans. Maxgel has been successfully used for embryoid body formation with human iPSCs4, cancer cells invasion or migration assays.

Hystem®: The HyStem platform is based on chemically synthesized hyaluronic acid, one major ECM component. Depending on the cell you are studying you can chose in the platform the best suited formulation: Hyaluronic acid alone (with crosslinker), or including collagen (GelinS) with or without Heparan Sulfate. Because it is a not a biological extract, it provide a better control of the composition of cells’ environment: control over growth factor incorporation, attachment factor incorporation, ECM protein incorporation, rigidity of the hydrogel. HyStem kits are optimal for culturing stem cells whose natural environments are rich in hyaluronic acid, but they have also been successfully used for several other applications like tissue engineering.

   HyStem Cell Culture

TrueGel3D biomimetic synthetic hydrogels: TrueGel3D™ hydrogels are biochemically defined hydrogels formed by mixing polymers with crosslinkers. They allow mimicking critical features of the natural extracellular matrix (ECM), including ECM proteins. They recapitulate native cellular environments similar to those of soft tissues, by supporting cell adhesion and protein sequestration. The TrueGel3D hydrogels platform offer the flexibility to choose between a predefined system or to decide whether you want to customize your cellular environment (gelation time, stiffness, bioactive component integration). It has been validated for several application, including cyst formation, coculture of cancer and stroma cell, or spheroids formation.

   TrueGel3D™

HydroMatrix Peptide Hydrogel (A6982): with his synthetic peptide nanofiber scaffold, HydroMatrix offers the precision and control of a synthesized matrix with the natural three-dimensional architecture of highly crosslinked peptide hydrogel. The HydroMatrix scaffold self-assembles from fluid precursors into a highly cross-linked peptide 3-dimensional hydrogel in response to changes in temperature or ionic strength. HydroMatrix promotes cell growth and migration and has been shown to support the proliferation of many cell types, including neural stem cells, neurons, glia, astrocytes, fibroblasts, and keratinocytes.

Hydromatrix has also been successfully used in combinaison with mesenchymal stem cell in wound healing5.

Materials

     

References

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  2. Kaisa Rajakylä,1 Ramaswamy Krishnan,2 and Sari Tojkander1,* Analysis of Contractility and Invasion Potential of Two Canine Mammary Tumor Cell Lines Front Vet Sci. 2017; 4: 149. Published online 2017 Sep 12. doi:  10.3389/fvets.2017.00149
  3. Legeay S1Billat PA1Clere N1Nesslany F2,3Bristeau S4Faure S1Mouvet C5. Two dechlorinated chlordecone derivatives formed by in situ chemical reduction are devoid of genotoxicity and mutagenicity and have lower proangiogenic properties compared to the parentcompound. Environ Sci Pollut Res Int. 2017 Feb 16. doi: 10.1007/s11356-017-8592-6.
  4. Songstad AE1Worthington KS1Chirco KR1Giacalone JC1Whitmore SS1Anfinson KR1Ochoa D1Cranston CM1Riker MJ1Neiman M2Stone EM1Mullins RF1Tucker BA1. Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced PluripotentStem Cell-Derived Choroidal Endothelium. Stem Cells Transl Med. 2017 Jun;6(6):1533-1546. doi: 10.1002/sctm.16-0399. Epub 2017 May 5.
  5. Fang S1Xu C2Zhang Y3Xue C1Yang C1Bi H1Qian X4Wu M5Ji K6Zhao Y6Wang Y7Liu H7Xing X8. Umbilical Cord-Derived Mesenchymal Stem Cell-Derived Exosomal MicroRNAs SuppressMyofibroblast Differentiation by Inhibiting the Transforming Growth Factor-β/SMAD2 PathwayDuring Wound Healing.
  6. Stem Cells Transl Med. 2016 Oct;5(10):1425-1439. Epub 2016 Jul 7.