Fabrication of thin‐layer matrigel‐based constructs for three‐dimensional cell culture

Abstract

Extracellular matrix‐based hydrogels such as Matrigel are easy‐to‐use, commercially available, and offer environments for three‐dimensional (3‐D) cell culture that mimic native tissue. However, manipulating small volumes of these materials to produce thin‐layer 3‐D culture systems suitable for analysis is difficult because of air–liquid‐substrate interfacial tension effects and evaporation. Here, we demonstrate two simple techniques that use standard liquid‐handling tools and nontreated 96‐well plates to produce uniform, thin‐layer constructs for 3‐D culture of cells in Matrigel. The first technique, the floating 3‐D cell culture method, uses phase‐separating polymers to form a barrier between the dispensed Matrigel, air, and cultureware surface to generate consistently thin hydrogels from volumes as low as 5 μL. These unanchored gels provide a useful assay for investigating airway smooth muscle cell contraction and may have future applications in studying asthma pathophysiology. The second technique, the fixed 3‐D cell culture method, provides an anchored gel system for culturing noncontractile cells (e.g., neurons) where 20 μL of Matrigel is dispensed into the bottom of a well filled with culture medium to form a thin gel containing embedded cells. This technique has potential widespread applications as an accessible 3‐D culture platform for high‐throughput production of disease models for evaluation of novel drug therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2733, 2019

SH‐SY5Y and LUHMES cells display differential sensitivity to MPP+, tunicamycin, and epoxomicin in 2D and 3D cell culture

“…Differentiated SH‐SY5Y cells offer a more stable source of neuron‐like cells, and 3D cell culture further offers the opportunity to capture both adherent and non‐adherent cell populations by embedding the cells inside a hydrogel matrix. The 3D culture technique described here offers an additional advantage by reducing the abundance of S‐type cells present in culture …”

“…SH‐SY5Y and LUHMES cells were cultured using standard 2D cell culture techniques or grown embedded within the growth factor reduced Matrigel (356230; Corning) Matrigel using our previously reported high‐throughput 3D culture method (3D) (Figure b,c) . An additional condition where 2D‐grown cells were covered with a layer of Matrigel (2D‐M) was also included to address the influence of mass transport differences between 2D‐ and 3D‐grown cells.…”

“…To address these limitations, we previously developed a simple, cost‐effective, and high‐throughput approach for the production of 3D neural cell cultures that minimizes cell attachment to culture plate surfaces . Using the SH‐SY5Y cell line, which has been used previously to model PD pathophysiology in vitro, we observed that cells displayed a more consistent neuronal phenotype following differentiation when grown in 3D Matrigel environments as compared to 2D culture .…”

“…To address these limitations, we previously developed a simple, cost‐effective, and high‐throughput approach for the production of 3D neural cell cultures that minimizes cell attachment to culture plate surfaces . Using the SH‐SY5Y cell line, which has been used previously to model PD pathophysiology in vitro, we observed that cells displayed a more consistent neuronal phenotype following differentiation when grown in 3D Matrigel environments as compared to 2D culture . Although the SH‐SY5Y cell line is widely used for toxicological assessment in the context of PD, there is debate as to its appropriateness as a model for DAergic neurons due to its limited expression of DAergic markers, mixed phenotypic expression of adherent and non‐adherent cells, and the presence of neuronal (N‐type) and substrate‐adherent (S‐type) epithelial‐like cells .…”

“…Moreover, only a few groups have attempted to characterize the growth of SH‐SY5Y and LUHMES cells in 3D culture, with no analyses conducted to explore the impact that 2D vs. 3D culture has on neurotoxicant sensitivity. Here, we used our technique for rapidly generating 3D matrigel‐based neural cell cultures to assess responses of the SH‐SY5Y and LUHMES cell lines to various toxicants previously used to model PD in vitro and in vivo: 1‐methyl‐4‐phenylpyridinium (MPP+), a selective DAergic neurotoxicant that induces mitochondrial dysfunction; tunicamycin (Tn), an ER stressor; and epoxomicin (Epox), a proteasome inhibitor. Responses to toxicants were assessed in 3D cell culture, conventional 2D cell culture, and in 2D‐cultured cells covered with a layer of Matrigel (2D‐M) to examine the influence of culture environment on cell phenotype and toxicant sensitivity.…”