Naked Liquid Marbles: A Robust Three-Dimensional Low-Volume Cell-Culturing System

Abstract

Three-dimensional (3D) multicellular structures allow cells to behave and interact with each other in a manner that mimics the in vivo environment. In recent years, many 3D cell culture methods have been developed with the goal of producing the most in vivo-like structures possible. Whilst strongly preferable to conventional cell culture, these approaches are often poorly reproducible, time-consuming, expensive, and labor-intensive and require specialized equipment. Here, we describe a novel 3D culture platform, which we have termed the naked liquid marble (NLM). Cells are cultured in a liquid drop (the NLM) in superhydrophobic-coated plates, which causes the cells to naturally form 3D structures. Inside the NLMs, cells are free to interact with each other, forming multiple 3D spheroids that are uniform in size and shape in less than 24 h. We showed that this system is highly reproducible, suitable for cell coculture, compound screening, and also compatible with laboratory automation systems. The low cost of production, small volume of each NLM, and production via automated liquid handling make this 3D cell-culturing system particularly suitable for high-throughput screening assays such as drug testing as well as numerous other cell-based research applications.

Neisseria meningitidis Induces Pathology-Associated Cellular and Molecular Changes in Trigeminal Schwann Cells

“…The cells exhibit similar cell-cell interactions to those in vivo, and, therefore, this culture system mimics the in vivo milieu better than 2D cell culture (31). Suspen-sions of single cells were seeded into naked liquid marbles and incubated overnight to allow the cells to form 3D spheroids, as previously described (31). The 3D cultures were then infected with N. meningitidis (MOI, 10:1) for 24 h and 72 h. Following incubation with bacteria, the response of trigeminal Schwann cells within the spheroids to N. meningitidis was examined using confocal microscopy.…”

“…We have developed a novel method for 3D cell culture termed the naked liquid (NLM) marble system, in which cells spontaneously and rapidly form 3D spheroid structures. The cells exhibit similar cell-cell interactions to those in vivo, and, therefore, this culture system mimics the in vivo milieu better than 2D cell culture (31). Suspen-sions of single cells were seeded into naked liquid marbles and incubated overnight to allow the cells to form 3D spheroids, as previously described (31).…”

Targeting Matrix Metalloproteinases: A Potential Strategy for Improving Cell Transplantation for Nervous System Repair

“…Scaffold-free methods employ low adherence conditions, such as using gravity-enforced techniques or super-hydrophobic surfaces for cells to form 3D spheroids or aggregates 15,[40][41][42] . Scaffold-free 3D culture methods can preserve cell-cell interactions and ECM production that mimic the in vivo microenvironment 15,43,44 and allow high cell densities 15,45 . Transplantation of MSCs in scaffold-free spheroids has shown promise for repair in rodents with SCI and PNI, respectively 46,47 .…”

The plant natural product 2-methoxy-1,4-naphthoquinone stimulates therapeutic neural repair properties of olfactory ensheathing cells

“…Three-dimensional (3D) culture and purification of primary mouse OECs. When cultured in our 3D Naked Liquid Marble (NLM) system 40 , mixed cultures of primary OECs and contaminating cells spontaneously self-organize so that DsRed-expressing OECs naturally separate from other cells (DsRed(−); primarily fibroblasts). Thus, the NLM system can be utilized to separate and purify cell populations.…”

“…Thus, the NLM system can be utilized to separate and purify cell populations. OECs were cultured in NLMs as previously described 40 . Briefly, cells were cultured in droplets (NLMs) on a superhydrophobic plate; here, cells rapidly spontaneously form uniformly sized spheroids inside the NLM.…”