Research Article: Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy

Date Published: January 25, 2017

Publisher: Public Library of Science

Author(s): Daniel Rodríguez-Martínez, María Magdalena Martínez-Losa, Manuel Alvarez-Dolado, Carlos E. Ambrósio.


Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE), a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80%) and yield (>70%). Post-thaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies.

Partial Text

Interneuron-related pathologies (interneuronopathies) comprise a wide and relevant group of diseases, including epilepsy, schizophrenia, infantile encephalopathies, autism spectrum disorder, or Alzheimer’s disease [1–5]. In the last years, different groups have been working in innovative cell-based therapeutic approaches to treat this group of neuropathologies [6–9]. Very promising results have been achieved grafting GABAergic interneuron precursors derived from the MGE, the subpallial region of the embryonic brain where most of the cortical interneurons are generated during development [10,11]. This therapeutic strategy has led to reversion of symptomatology in multiple animal models of the above mentioned diseases [6–9].

In order to establish the best cryopreservation conditions for MGE-derived GABAergic precursors we designed four different experimental procedures. We assessed how the physical structure (in toto explants, NS, or dissociated cells) affected the cell viability of frozen MGE-derived precursors. In addition, we studied the influence of an in vitro amplification step on their survival and features. Detailed description of the four experimental protocols can be found in materials and methods section (Fig 1).

Neuronal precursor transplantation is a promising therapeutic strategy for brain repair and treatment of diverse neuropathologies. The accessibility to a permanent source of neuronal progenitors is essential for the clinical application of these cell-based therapies. Biobanks, in where to cryopreserve tissue or neuronal precursors, can meet this need as reliable and constant cell suppliers [39]. However, appropriate cryopreservation protocols are necessary to minimize the effects on cell viability and ensure the correct neuronal progenitor storage. In addition, it may be convenient an amplification of the precursors to increase their availability, keeping always intact their intrinsic properties of migration, proliferation, and differentiation.