Product Survey: 3D CEll Culture Systems

The World isn’t Flat
by Harald Zähringer, Labtimes 01/2016

Ross Harrison’s hangig drop frog nerve cell cultures were the first attempts at 3D cell culture. Photo: digital HPS

Cells are embedded under natural conditions into an extracellular matrix, which allows them to communicate with each other and to grow into all three dimensions. 3D cell culture systems are aimed at mimicking the meshwork of the matrix and support three dimensional propagation of the cells.

The American anatomist and embryologist Ross Granville Harrison was one of the first life scientists to experiment with cell cultures early in the 20th century, already. To study the development of frog nerve cells in his laboratory at the University of Yale, Harrison suspended explants from frog neural tubes in a drop of frog lymph, sitting on a sterile cover slide. He inverted the cover slide with the clotted lymph over a glass depression slide to obtain a hanging drop culture system that enabled him to observe the growing neurons.

It once started in 3D

Harrison’s modified hanging drop method was not only one of the earliest attempts to cell culture, in general – it was also one of the pioneering experiments of 3D cell culture. Similar to their natural environment in frog tissue, the nerve cells were able to establish three-dimensional cell-to-cell contacts and grew in all three dimensions.

But Harrison’s hanging drop method had a few downsides, which hampered the handling and maintenance of the cultured cells. Changing the cell culture medium of the hanging drop was one of its major issues; dividing and subculturing the cells, another.

Hence, most researchers shied away from Harrison’s pioneering 3D cell culture technique and developed easier-to-handle, two-dimensional cell culture models, e.g. adherent cells growing on the surfaces of culture dishes and microplates, or suspension cultures raised in bottles filled with cell culture media. Both techniques force the cells to grow in two dimensions with only lateral contacts, or even worse, as complete mavericks swimming around in the cell culture media. Hence, adherent or suspension cell culture may facilitate cell culture routines, such as media exchange and cell divisions, but they are only a poor copy of the cellular in vivo environment.

More than just a framework

It was 3D cell culture pioneer Mina Bisell from the Lawrence Berkeley National Laboratory, USA, who pointed out in the early nineteen-eighties that cells are embedded under natural conditions in an extracellular matrix (ECM) consisting of proteoglycans and fibrous proteins, including collagen, elastin, fibronectin and laminin. The ECM-meshwork stabilises the structure of the tissue and establishes the three-dimensional form of the cell network. But the ECM is more than just a framework for the cells: it directly effects their development, migration profile, shape and function as well as their communication via growth factors and other secreted cell signals.

Since most of the crucial functions of the ECM are completely missing in 2D cell culture models, more and more researchers try to establish 3D cell culture systems, in order to imitate the ECM and to get closer to in vivo conditions. They usually favour 3D cell culture approaches based on spheroids, scaffolds or gels.

Spheroids are globular cell clusters formed by aggregating adherent cells, which were first recognised by the German embryologist, Johannes Holtfreter, during his work with embryonic cells in 1944. As spheroids organise their own ECM during growth, they may be generated without the need of special scaffolds or matrices, by a simple hanging drop technique resembling Harrison’s early approach.

Spheroids in hanging drops

Instead of depression slides and cover slides made of glass, as in Harrison’s pioneering work, special plastic microplates are usually applied today, to create spheroids. Hanging drop plates resemble typical microplates with one exception: they come with a little puncture in every access hole. Small volumes of suspended cells are pipetted into the holes on top of the plate. Driven by gravity, the cell suspension then seeps through the holes and forms hanging drops on the bottom of the plate facilitating spheroid generation.

The formed spheroids are similar in shape and structure to solid tissues and tumours, and lend themselves as models in drug delivery or tumour studies. Due to the lacking scaffold and the fragile inherent ECM, however, spheroids are prone to necrosis when they reach a critical diameter of approximately 500 to 600 micrometres.

Scaffolds, also called 3D matrices, are probably the most widely used 3D cell culture systems. They are available in countless different materials and in diverse formats but they all share the same purpose: scaffolds mimic the ECM and provide a physical support to the growing cells. Besides that, they may also force cells into a specific geometric structure during propagation.

Natural or artificial

Based on the materials used, 3D cell culture scaffolds can be roughly classified into the categories biological and synthetic. Typical synthetic scaffold materials are metals, such as tantalum, magnesium, titanium and their respective alloys as well as ceramics and bioactive glasses made, for example, from hydroxyapatite or calcium phosphate glass.

Due to their inertness, lacking surface activity and rigid structure, the above-mentioned metals are predestined as implanting materials, which may also be coated with collagen or fibronectin to support in vivo bone formation.

Porous structures

Ceramics and bioactive glasses, on the other hand, show a porous microarchitecture, which may be effectively infiltrated by osteoblasts and other cells during proliferation and differentiation. Furthermore, these materials are bioactive, osteoconductive, i.e. facilitate bone repair and are able to bind directly to bone.

But especially bioactive glasses have even more to offer to tissue and bone “engineers”: they may also stimulate intrinsic cell responses leading to osteoconductive “behaviour” of the cells, bind not only to bones but also to soft tissue, and may carry metal ions and bioinorganic compounds. As a further bonus, they are easily degraded by the organism, without harmful effects when they have accomplished their mission as 3D cell culture scaffold.

Flabby gels

Gels are soft, tissue-like scaffolds, which mimic the ECM. They are either derived from natural sources, e.g. the Engelbreth-Holm-Swarm mouse sarcoma, which is full of ECM proteins, such as collagen and laminin or are made of synthetic polymers.

The properties of natural gels come pretty close to the ECM requirements but they have a few drawbacks as well. Batch-to-batch variation is one of the major issues with natural gels, harbouring undefined and unwanted substances, for example, animal viruses, or residual growth factors being another. The flabby consistence of 3D cell culture gels makes things even worse – some must be kept on ice to enable proper handling.

Modified properties

To avoid these problems, many researchers rely on synthetic gels based, for example, on modified polyethylen glycol, hyaluronic acid or polycaprolactone. Synthetic gels are free of unspecified substances and come with predictable mechanical and physical properties.

Choosing an ideal spheroid-, scaffold- or gel-based 3D cell culture system for a certain cell type or experimental set-up may cause quite a headache. The survey of available 3D cell culture systems on the next pages should support the decision-making.

First published in Labtimes 01/2016. We give no guarantee and assume no liability for article and PDF-download.

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