For decades, three-dimensional (3D) cell culture has been employed by tissue engineers, stem cell scientists, cancer researchers and cell biologists, largely in university settings. The development of new materials or methods has been driven by the desire of these scientists to incorporate experimental systems that better represent the in vivo environment into their research. Early adopters of 3D cell culture technology have reaped the benefits of better data with groundbreaking knowledge of tissue and cancer behavior.
3D cell culture methods were once expensive, messy, laborious, and difficult to adapt to existing procedures. Today, researchers can pick among an array of 3D cell culture tools, ranging from simple to complex, to fit their specific needs. Segmentation of the 3D cell culture choices into discrete categories demonstrates a new maturation in the market. As the number of 3D cell culture options has grown, publications proving their importance have exploded in numbers (Figure 1).
Figure 1. Publications referencing 3D cell culture have grown dramatically in the last several years. Each line on the graph represents a separate search for threedimensional cell culture plus the additional listed search term. A small degree of overlap is possible.
If you are new to 3D, what do you choose?
An important consideration in 3D cell culture is replicating or mimicking the extracellular matrix (ECM) (Figure 2). The ECM provides physical structure, sequesters and secretes growth factors, and facilitates cellular communication. Consideration of the ECM composition, structure, and density in your target tissue, or whether or not you would like to provide ECM at all, may dictate the 3D cell culture format choice.
In this tutorial, we discuss 3D cell culture methods that rely on cells to secrete their own ECM and others that utilize natural or artificial materials to mimic the ECM until cells create their own. Five types of 3D cell culture options are reviewed: scaffold-free platforms for spheroid growth, scaffolds, gels, bioreactors, and microchips. For your convenience, select review articles are included at the end of each section.
Figure 2. Schematic of various components of the ECM.
Spheroids are self-assembled spherical clusters of cell colonies. They were first documented in 1944 by Johannes Holtfreter who worked with spherical aggregates of embryonic cells. Spheroids naturally mimic solid tissues, avascular tumors, and embryoid bodies, and have found application among researchers in cancer and stem cell research. With inherent metabolic (oxygen, carbon dioxide, nutrients, wastes) and proliferative gradients, spheroids serve as excellent physiologic models.
Scaffold-free platforms for spheroid growth do not contain added biomaterials or ECM, and cells grown in them generate and organize their own 3D ECM, so spheroids closely resemble in vivo tissues. Co-cultures with other cell types (i.e., endothelial, stromal, epithelial cells) extend the predictive cytotoxicity capabilities of this 3D cell culture system.
Scaffold-free platforms have no support structure or porosity. The overall spheroid size is limited beyond a critical size of 500 – 600 μm in diameter, after which central secondary necrosis develops in most, but not all, spheroids grown from permanently-transformed cell lines.
Standardized mass production of 3D spheroids makes them applicable for both basic laboratory research and high-throughput screening (HTS) applications. The Perfecta3D® Hanging Drop Plate from 3D Biomatrix™ is designed to enable consistent formation of spheroids using conventional liquid handling tools. This scaffold-free platform is simple to use and generates spheroids with consistent sizes and shapes so that testing is controllable and reliable. Adjusting the seeding density, from as few as 50 cells to as many as 15,000 cells, allows production of varying spheroid sizes.
The plate consists of the main hanging drop culture plate and a complementary lid and tray, which serve to maintain sterility and reduce evaporation. Access holes in the culture plate allow manipulation of fluids and spheroids from the topside. A water reservoir constructed around the periphery of the culture plate also helps to alleviate evaporation (Figure 3).
Figure 3. A schematic of the Perfecta3D Hanging Drop Plate.
Hanging drops are created by dispensing small volumes of cell suspensions, using standard pipette tips, into the access holes on the top of the plate, just like pipetting into conventional multi-well plates. In a similar fashion, reagents and drugs can be added to or removed from each hanging drop.
A plateau structure on the bottom of the plate stabilizes the hanging drops (Figure 4). The Perfecta3D Hanging Drop Plates do not have a bottom substrate for cells to eventually attach to, therefore cells in suspension aggregate into a spheroid.
Figure 4. Spheroids are created by dispensing cell suspensions into the access holes of the Perfecta3D Hanging Drop Plate, just like pipetting into conventional multi-well plates.
Spheroids can be harvested and analyzed using colorimetric, fluorescence, and luminescence assays measured with a plate reader. Microscopic imaging of spheroids can be performed directly with the transparent plate, lid and tray assembled. The platform also offers simplified liquid handling procedures and compatibility with HTS instruments, such as liquid handling robots like the Biomek® FX and epMotion automated pipetting systems.
Articles:
The inventing University of Michigan research team discusses the Perfecta3D Hanging Drop Plate.
Demonstration of Z-factors and co-cultures with the Perfecta3D Hanging Drop Plate.
An overview of 3D in vitro cancer models as they pertain to drug discovery, with a specific focus on those that have been developed from a tissue engineering perspective.
Scaffolds, also commonly called 3D matrices, are available in a large variety of materials with different porosities, permeabilities and mechanical characteristics designed to reflect the in vivo ECM of the specific tissues being modeled. Scaffolds are manufactured using a variety of techniques, such as 3D printing, particulate leaching, or electrospinning, each of which introduces different porosities, pore sizes, scaffold materials and features.
Scaffolds are typically divided into two main application categories: functional implants for clinical and regenerative medicine applications and in vitro 3D scaffolds for laboratory applications.
Though the characteristics of implantable scaffolds may vary greatly depending on the tissue being mimicked, the ultimate goal for many implantable scaffolds is to provide support to a wound site and aid eventual replacement of the scaffold by natural tissue. As such, the requirements for functional implant scaffolds differ from those for in vitro 3D laboratory applications. Functional implants must match the defect site, support and promote desired cell growth, and biodegrade without harmful effects.
When scaffolds are used for in vitro laboratory applications, geometric match and biodegradability are less necessary. In fact, degradability may introduce an undesirable variable into experiments as byproducts may change the chemistry and pH of the culture system. Furthermore, as the scaffold degrades and cells re-organize the matrix, the cells may not retain their 3D configuration. In vitro scaffolds should represent a more stable structure and function similar to the natural in vivo environment.
3D scaffolds for in vitro laboratory applications are available in a variety of materials: metals, ceramics, polymers - natural and synthetic, and composites. Properties to consider include biocompatibility, wettability, mechanical properties, and surface chemistry. The method of fabrication must also be considered as it may introduce a random or ordered structure to the scaffold (Figure 5). When utilizing a scaffold with a random structure, such as those that result from particulate leaching methods, scaffold-toscaffold variability, as well as isolation of areas within the scaffold, may be a problem.
Figure 5. Examples of random (left) and ordered (right) scaffold structures. Image adapted from Lee et al.
Due to the variety of material and structural choices for scaffolds, they are widely used in many applications. Furthermore, as they provide a surface on which cells can grow, they can easily impart 3D growth with little alteration to cell culture procedures. The porosity of scaffolds aids mass transport of nutrients, oxygen, and wastes, allowing for larger culture growth than the scaffold-free platform discussed earlier; it can be difficult to extract all cells for analysis with increased scaffold size and tortuosity. Imaging may also become difficult depending on the scaffold size, transparency of material, and depth of the microscope.
In a 2008 review article, Lee et al. discuss the macro-, micro-and nano-scale elements of 3D scaffolds.
Articles:
A review of 3D cell-growth techniques and scaffolds analyzed from the perspective of materials properties, manufacturing and functionality.
General review of 3D cell culture approaches and techniques.
