Organoid EP – with the NEPA21 System
We have had considerable success with the NEPA21 system for Organoid EP.
Methodology and Publications
– Modelling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids
o Toshiro Sato, et al – June 2014
o In this paper, the authors dissociated human colonic organoids into single cells and electroporated using the NEPA21 and cuvettes
o RESULTS: Liver Organoid Results – Cuvette EP (with the NEPA21 and our EC-002S Cuvettes)
– Efficient genetic engineering of human intestinal organoids using electroporation
o Toshiro Sato, et al – September 2015
o This paper is an optimisation of the previously developed organoid culture protocol presented in the earlier paper
o In this paper, the authors describe a method for modulating genes of interest in untransformed human colonic organoids via electroporation of gene vectors.
o This paper presents a detailed (step-by-step) protocol for the generation of intestinal organoids by culture with essential growth factors in a basement membrane matrix.
o The paper also describes how to stably integrate genes via the piggyback transposon, as well as precise genome editing using the crispr-cas9 system.
o Beginning with crypt isolation from a human colon sample, genetically modified organoids can be obtained in 3 week. Isolated intestinal crypts to be expanded for over a year with the addition of essential growth factors and after embedding into basement membrane matrix (Matrigel)
o Under the described culture conditions, the intestinal stem cells organize into 3D crypt-villus structures named intestinal epithelial organoids. This provides a source of untransformed human intestinal cells that are independent of pre-existing cell lines
o Comparative evaluation of various gene delivery methods, showed that the electroporation method, combined with several modifications to the culture conditions, yielded relatively high transduction efficiency compared with liposomal transfection or gene electrotransfer using nucleofector
o An additional advantage of the presented electroporation method is that genes can be transferred upon preparation of the plasmids, which alleviates the labour and time that are required to produce lentivirus or transgenic rodents
The above papers use:
– the NEPA21 electroporator
– our EC-002S cuvettes
– CU500 Cuvette Chamber and
– CU600 Cuvette Stand
Further Organoid Information
Clients report successful electroporation of organoids in matrigel drops.
We have sold clients the following electrode configurations to support their wider organoid research:
– CUY701P10E and CUY701P7L
o The methodology used for this electrode combination is similar to the method recommended for Brain Slice EP