Over the past decade, biomedical scientists and engineers have increasingly abandoned conventional cell culture methods, which often confine cells to a monolayer at the bottom of a dish or plate, in favor of novel 3D techniques.
A number of presentations at the “3D Cell Culture” conference, held in Zurich earlier this year, and sponsored by the DECHEMA Society for Chemical Engineering, revealed the current state of the art—and what is on the horizon.
“By introducing more organotypic, more physiologic model systems, scientists will gain higher-quality data assessment and better ‘de-risking’ of their compounds in an in vitro situation before they start with expensive preclinical and clinical trials,” explained Jens Kelm, Ph.D., cofounder of InSphero.
“The idea of developing more complex model systems in vitro is to improve the functionality of the cells to mimic more closely what is going on in the human body or in the animal.”
The first model system developed by InSphero is that of spherical tumor microtissues, which are thought to behave similarly to tumors. These spheroid microtissues self-assemble from cells in hanging drops, even in the absence of scaffolds. Dr. Kelm noted that hanging-drop technology is not itself new; however, advances in automation and equipment now allow formation of microtissues in 96-well plates, where they may easily be assayed for properties such as sensitivity to drugs.
Having demonstrated that tumor microtissues can be created and assayed in high-throughput fashion, Dr. Kelm’s team is now working toward additional scaffold-free models for the four main organs of toxicology research: brain, heart, kidney, and liver. In each case, models will be considered valid if they can perform tissue-specific functions.
For tumors, these functions include expression of cell-surface receptors and formation of extracellular matrix (ECM), said Dr. Kelm.
“For the liver, it’s the metabolic capacity and detoxification capacity of the hepatocytes, as demonstrated for InSphero’s recently launched rat and human liver microtissue models. Similarly, for the kidney cells, you would look primarily at the transporters of the epithelial cells. The heart is one of the easiest tissues to assess—you look at the contraction of the tissue over time.”
Stefan Przyborski, Ph.D., CSO of Reinnervate, addressed the topic of scaffolds for 3D cultures. His research, like that of Dr. Kelm, grew out of concerns that cells grown as a monolayer did not behave like cells in vivo. Not only do these cells become unnaturally elongated and flattened, they are under stress and have limited potential for cell-cell interactions.
Reinnervate aims to facilitate growth of cultures in 3D with a scaffolding material that it calls alvetex®. Alvetex is made from polystyrene, as is 2D cell culture plasticware, but comes as a highly porous 200-micron-thick membrane into which cells can grow and maintain their natural 3D structure.
“Alvetex polystyrene scaffold simply provides cells with a support structure that allows them to move inside and to continue to grow, while not allowing them to flatten out, as would happen in a traditional 2D culture setting,” according to Dr. Przyborski.
“Many cells find the alvetex scaffold environment very suitable, and start to lay down their own ECM proteins as they would in vivo. For some cell types, like primary hepatocytes, there may need to be an ECM coating on the alvetex scaffold to help establish the 3D culture.”
Thin and Porous
Because of the thinness and porosity of scaffolds such as alvetex, cells inside the scaffold may exchange nutrients and waste products with the growth media via passive diffusion. Media is typically replaced by routine manual pipetting, though a new collaboration between Reinnervate and Tecan will work toward automation of this process.
The best placement of a scaffold within the wells of a plate depends on the downstream assays that are planned.
“Certain techniques like transfection may work better with plate formats, in which the scaffold is at the bottom of wells,” noted Dr. Przyboski, “whereas assays such as cell invasion may be better suited to well insert formats, where the scaffold is suspended in the media. Most cell types will grow in either format, the difference being that usually the well insert format promotes growth deeper into the scaffold in the same time frame.”
Thomas Weiser, D.V.M., Ph.D., global head of early and investigative safety for Roche, also touted the promise of scaffolded 3D cultures. The goal of Dr. Weiser’s work at Roche is “to reduce and refine the use of experimental animals by well-characterized and validated in vitro models,” he said. “We believe that 3D models hold the most promise to achieve this goal.”
Dr. Weiser reported on a 3D liver model based on RegeneMed technology, in which nonparenchymal cells (Kupffer cells, endothelial cells, stellate cells, and bile duct epithelial cells) are grown on a nylon scaffold, followed by addition of hepatocytes.
“We are also looking at other technologies,” he stated, “as they are all in a rather early stage, and it needs to be shown which one is best suited with regard to robustness, throughput, et cetera.”
Roche’s preliminary data suggests that the nonparenchymal cells are needed for in vitro reproduction of in vivo responses to model compounds. 3D co-cultures may thus enable studies of the mechanisms underlying these responses.