Cardosins: A new and efficient plant enzymatic tool to dissociate neuronal cells for the establishment of cell cultures

Abstract

In the present work, we examined the feasibility of using cardosins, plant aspartic-proteinases from Cynara cardunculus L., to isolate cells from rat embryonic brain. Using morphological and functional assays, we compared cell cultures obtained with cardosins with those prepared with a well-established trypsin protocol. Cardosins and trypsin dissociation produced cells with similar yield, viability, and GABA release in response to a depolarizing stimulus. However, cardosins-dissociated cells appeared to recover faster in culture, as assessed by the MTT-test and by the number and length of neurtites, suggesting that cardosins are less aggressive to neurons than trypsin. This feature might be helpful for research and medical purposes requiring fast manipulations of cells.

Cardosins improve neuronal regeneration after cell disruption: a comparative expression study

“…Actually, as shown in Fig. 1, neurons isolated with cardosins and cultured for 24 h after plating consistently exhibited longer neurites (50±6.0 mm, P≤ 0.01), compared to the cultures of cells dissociated with trypsin (35.0±4.0 mm, P≤0.01), but the differences were no longer apparent in cells cultured for longer periods of time (Duarte et al 2007). …”

“…These neurons exhibit normal morphology and function when compared to cultures prepared by the widely used trypsin dissociation method. This new protocol, when compared to the classic protocol, shows an improvement on neuronal recovery after cell plating, suggesting that cardosin dissociation of brain tissue is less aggressive to neurons than trypsin (Duarte et al 2007). As a matter of fact, we showed that cells isolated with cardosins appear to recover faster than those isolated with trypsin, as assessed by the number and length of neurites.…”

“…Cell isolation and culture Primary cultures of brain cortical cells were prepared from Wistar rat embryos (E15-E16) as described by Duarte et al (2007). Animals were handled following the approved guidelines of national ethical requirements for animal research and in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes.…”

“…In a previous work we reported that cardosins can be used to dissociate embryonic cortex to prepare neuronal cultures exhibiting normal morphology and function compared to cultures prepared by the widely used trypsin dissociation (Duarte et al 2007). Actually, as shown in Fig.…”

“…Their maximal length was 35.0± 4.0 mm in trypsin-generated cultures, and 50±6.0 mm (P≤0.01) in cultures obtained with cardosins. No differences were perceptible after 7 days in culture (Duarte et al 2007). These aspartic proteinases exhibit a narrow specificity cleaving peptide bonds between hydrophobic amino acids (Sarmento et al 2004).…”