Novel culture strategy for human stem cell proliferation and neuronal differentiation

Abstract

Embryonal carcinoma (EC) stem cells derived from germ cell tumors closely resemble embryonic stem (ES) cells and are valuable tools for the study of embryogenesis. Human pluripotent NT2 cell line, derived from a teratocarcinoma, can be induced to differentiate into neurons (NT2-N) after retinoic acid treatment. To realize the full potential of stem cells, developing in vitro methods for stem cell proliferation and differentiation is a key challenge. Herein, a novel culture strategy for NT2 neuronal differentiation was developed to expand NT2-N neurons, reduce the time required for the differentiation process, and increase the final yields of NT2-N neurons. NT2 cells were cultured as 3D cell aggregates ("neurospheres") in the presence of retinoic acid, using small-scale stirred bioreactors; it was possible to obtain a homogeneous neurosphere population, which can be transferred for further neuronal selection onto coated surfaces. This culturing strategy yields higher amounts of NT2-N neurons with increased purity compared with the amounts routinely obtained with static cultures. Moreover, mechanical and enzymatic methods for neurosphere dissociation were evaluated for their ability to recover neurons, trypsin digestion yielding the best results. Nevertheless, the highest recoveries were obtained when neurospheres were collected directly to treated surfaces without dissociation steps. This novel culture strategy allows drastic improvement in the neuronal differentiation efficiency of NT2 cells, insofar as a fourfold increase was obtained, reducing simultaneously the time needed for the differentiation process. The culture method described herein ensures efficient, reproducible, and scaleable ES cell proliferation and differentiation, contributing to the usefulness of stem cell bioengineering.

Cryopreservation of Adherent Cells: Strategies to Improve Cell Viability and Function After Thawing

“…Cells adherent to glass coverslips were processed as described previously. 47 Primary antibodies and dilutions used were anti-tubulin beta III isoform (b-tubulin, 1:10; Chemicon, Temecula, CA) and anti-microtubule-associated protein-2 (MAP2a=b, 1:100; Sigma). The secondary antibodies and dilutions used were anti-mouse Alexa 488 (1:500) and anti-rabbit Alexa 594 (1:500) (Molecular Probes, Eugene, OR).…”

Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method

“…Neurosphere dissociations are commonly performed by using enzymatic methods which can generate high yields of single dissociated cells with reproducible results. However, to avoid protease and collagenolytic activities, which degrade cell membrane proteins [3], mechanical methods including trituration [4], manual cutting with scalpel [5], micro-scissors [6], tissue chopper [7], and microfabricated filter are used [8]. We have developed a microfluidic chip-based mechanical method to dissociate neurospheres [9].…”

A microchip for quantitative analysis of CNS axon growth under localized biomolecular treatments

“…To enhance the survival and growth of neurons in vitro , culture substrates such as glass coverslips or multi-well culture plates are often coated with extracellular matrices (ECMs) (Baron-Van Evercooren et al , 1982; Gingras et al , 2008; Serra et al , 2007; Taylor et al , 2007; Vyas et al , 2010). However, the direct effect of ECMs on axon growth has not been fully characterized.…”