Abstract
Many extracellular matrices (ECM) used for modern cell culture are derived from animals. An alternative approach is the recombinant production of individual matrix protein components. A further development of this strategy uses a constant core protein polymer that is modifiable with functional domains of various ECM proteins. This way, a single, highly defined ECM system could be used for a large variety of cell types. Self-assembling protein domains from human muscle sarcomeres, termed here ZT material (ZT), have been shown to be suitable for this modular approach of generating ECMs. We explored in a proof-of-concept study, whether ZT, modified with the fibronectin 10 domain (ZTFn10) is able to substitute bovine serum-derived fibronectin as coating for neural crest cell (NCC)-based toxicity testing. Human NCC were generated from pluripotent stem cells and used in the automated version of a NCC migration assay (cMINC). ZTFn10, but not the unmodified core material (ZT), allowed for a high migration activity. The classical cMINC setup, with bovine fibronectin coating, was used as positive control, and detailed analysis of NCC migration by time-lapse recording indicated that the novel ECM fully matched the bioactivity of the traditional ECM. A final set of experiments showed that various positive controls of the cMINC assay (PCB180, LiCl, cytochalasin D) showed nearly identical inhibition curves on the traditional and the novel ECM. Thus, the cMINC, and possibly other bioassays, can be performed with a ZT-based ECM instead of traditional animal-derived protein coatings.
Highlights
While cell culture experiments have successfully replaced several animal studies, many in vitro methods still use animal-derived materials
3.1 Migration of neural crest cells on the ZT modified with the fibronectin 10 domain (ZTFn10) polymer In order to investigate whether the ZTFn10 biomaterial may substitute the established bovine serum Fn coating in the circular migration inhibition of neural crest cells (cMINC) assay setup, we compared the migration of NCCs on the novel Z1212/telethonin polymer (ZT) matrix to that on the classical coating
The polymers were passively adsorbed onto polystyrene 96-well cell culture plates, and NCCs were plated on the coated surfaces
Summary
While cell culture experiments have successfully replaced several animal studies, many in vitro methods still use animal-derived materials. The search for chemically-defined and non-animal sourced cell culture material has become a research priority in its own right. The goal is to make in vitro systems more reproducible (independent of lot effects) and to avoid ethically problematic procedures currently used to produce cell culture medium supplements, extracellular matrix, and analytical tools such as animal-derived antibodies. Serumfree media are in routine use only for a few cell types, such as LUHMES cells (Scholz et al, 2011) or induced pluripotent stem cells (iPSCs) (Hackland et al, 2017; Reichman et al, 2017; Sung et al, 2019), but no universal, chemically-defined, serum-free culture medium is available (van der Valk et al, 2018). The FCSfree database was launched to increase the use of serum-free
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