Methods and Protocols (5): A New Coating Compound
(September 25th, 2014) Coating glass coverslips with fibronectin or poly-L-lysine can be expensive or uncomfortable for the cells. Italian researchers have synthesised a new, biocompatible “cell glue” and tested it on primary brain cells, with success.
Growing primary brain cells on coverslips isn’t an easy task. The most basic prerequisite is appropriate coverslip surface-coating because, without a proper fundament, neurons and glia cells simply won’t attach to their new unnatural surrounding and, hence, won't grow, spread and differentiate. To make their cells stick, researchers use extracellular matrix substances like fibronectin or synthetic polypeptides such as poly-L-lysine as “adhesion promoters”. But, in some cases, these molecules are cytotoxic and require multiple washing steps before cell seeding. Or they are expensive to produce or purchase.
Recently, Italian researchers tested an alternative coating chemical called AGMA1, an amphoteric, water-soluble polyamidoamine. This new synthetic adhesion molecule is reportedly less toxic than poly-L-Lysine (PLL) and also much cheaper than fibronectin. AGMA1 can, more or less, be easily prepared by adding Agmatine sulfate and lithium hydroxide monohydrate to a solution of 2,2-bisacrylamidoacecetic acid and lithium hydroxide monohydrate in distilled water. The full protocol can be accessed here.
But can it live up to its full adhesion potential? To find out, Paolo Ferruti from Milan University and his colleagues prepared stock solutions of poly-L-lysine, poly-L-ornithine hydrobromide and AGMA1 in milli-Q sterile water, filtered them through a 0.22 μm filter, diluted their solutions to 1 mg/ml and coated a few glass coverslips by “aseptically dispensing 200 ul adhesion promoter solution”. Next, they prepared primary cell cultures from 18-day-old rat embryos (neuronal cultures) and embryonic rat pups at E21 (hippocampal neural and astrocytic cultures) and seeded the cells onto the coverslips. The result? AGMA1 did just as well as the cytotoxic poly-L-lysine and poly-L-ornithine. “Microglia cells cultured on AGMA1-coated coverslips substrate displayed the typical resting, ramified morphology of those cultured on poly-L-lysine and poly-L-ornithine (...). Mixed cultures of primary astrocytes and neuronal cells grown on AGMA1- and poly-L-lysine coated coverslips were morphologically undistinguishable. On both substrates, neurons differentiated axon and dendrites and eventually established perfectly functional synaptic contacts.”
Being cheaper and less cytotoxic in vitro are not all AGMA1’s advantages. AGMA1 is biocompatible and thus, in hydrogel form, might be suitable as a scaffold for tissue engineering purposes, such as peripheral nerve regeneration. According to Ferruti, polyamidoamines such as AGMA1 “are highly versatile functional polymers, whose biotechnological applications are still waiting to be fully exploited.”
Nicola Hunt & KG
Image: Chris Schlag