Usage of 3D Cell Culture

Introduction

The usage of three-dimensional (3D) cell culture in microfluidic devices was first published in 2009 in a collaborative study from MIT. It had already been established that 3D cell cultures were considerably better than 2D cell cultures due to their inclusion of the extracellular matrix (ECM) and other growth factors. Upon this initial study, it was found that the formation of these 3D spheroids showed more accurate growth pattern and treatment results compared to their 2D counterpart. However, a different set of considerations must occur for 3D cell culture, such as the materials used and unintentional oxidation. (1)

These devices are typically made with PDMS consisting of several different micropillars. These micropillars are evenly spaced such that there is an increase of cell-to-cell interactions and a decrease in physical shear stress. (2) PDMS is a material that has always been considered the norm for microfluidic devices and 2D cell culture. As such, to prove the 3D model, PDMS devices were also fabricated with these microarrays. These devices consist of several inlet and outlet streams for the injection of cells and removal of byproducts, respectively. In addition, several different methods of cellular loading exist depending on the cell type (3). The hanging drop method is ideal for total loading of 50 uL at a rate of 5 uL/min. This allows for the cells to be tightly packed in each drop without breaking formation held together from surface tension. The forced-floating method allows for larger long-term loading with an increase of cell-to-cell interaction. However, an agarose gel or some other scaffold must be coated in the bottom of the wells and subsequently incubated for 24 hours at 37C. As such, the cells must have a longer doubling time in order for proper experiments can occur for adding any form of therapeutic. Another method for loading cells onto the microfluidic device involves directed scaffolds with growth factors, such as collagen IV attached. This would allow for continuous cell growth after several passages (3).

Usage of PDMS vs. Polystyrene

One of the current issues with PDMS is the interaction of biomolecules with the surrounding material. This can impact cell proliferation due to the absorbance of growth factors from the scaffold and interference with the cell culture media. In addition, in the case of therapeutics testing, this can skew the results such that the therapeutic can be absorbed into the surrounding material rather than target the cells. From a design standpoint, a polystyrene based microfluidic doesn’t require micropillars as PDMS does. This is due to polystyrene’s interconnected nature and hydrophobicity with interaction between wells. The material will not allow for passage through of culture media, therefore cells would not be able to travel between the walls of wells either. However at this time, it is unknown how long the cells can survive in this device as the current study only shows a 4-day long experiment. But it does provide the possibility that polystyrene would be able to determine different gas testing abilities, such as with cigarette smoke. (4)

Air Bubble Formation

One of the current challenges with 3D spheroid formation is air bubble formation. One of the problems with air bubbles is the increased amount of oxygen within the cell culture which will lead to reduction in stability and flow resistance in the normally Nitrogen environment. Different culture flows were tested at media concentrations ranging from 10% to 40% with these endpoints providing the lowest amount of bubble formation. (5)

https://www.fasebj.org/doi/pdf/10.1096/fj.08-122820. https://ac.els-cdn.com/S1872204017610296/1-s2.0-S1872204017610296-main.pdf?_tid=281fb307-8033-4c4c-acee-4ef301fc0d0b&acdnat=1551062202_0b4f2ab2b7924f06c9adc1d2a285b604 Microfluidics-based 3D cell culture models: Utility in novel drug discovery and delivery research A polystyrene-based microfluidic device with threedimensional interconnected microporous walls for perfusion cell culture Development of a microfluidic perfusion 3D cell culture system A novel 3D mammalian cell perfusion-culture system in microfluidic channels