Freshly frozen E18 rat cortical cells can generate functional neural networks after standard cryopreservation and thawing procedures

Abstract

Primary dissociated brain tissue from rodents is widely used in a variety of different scientific methods to investigate cellular processes in vitro. Often, for this purpose cell cultures need to be generated just on time, requiring extensive animal lab infrastructure. We show here that cryopreservation and thawing of dissociated tissue from rat cerebral cortex at embryonic day 18 is feasible without affecting its ability to form functional neuronal networks in vitro. Vitality of fresh and re-thawed cortical cells was comparable, assessed by CellTiter-Blue-assay, CytoTox-ONE assay, immunocytochemical characterization and in vitro neuronal network activity recordings on microelectrode arrays. These findings suggest that planning and execution of experiments might be considerably facilitated by using cryo-preserved neurons instead of acutely dissociated neural cultures due to fewer logistical issues with regard to animal breeding and pregnancy timed preparations.

High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons

“…Using whole-cell patch clamp electrophysiology, we observed similar examples of spontaneous and evoked APs elicited by fresh and cryostored neurons, in agreement with previous reports that the number, burst duration, and amplitude of APs are not significantly affected following cryostorage in several media ( Otto et al, 2003 ; Quasthoff et al, 2014 ; Pischedda et al, 2018 ). Additionally, careful analysis of specific features of evoked AP waveforms did not detect significant differences after recovery from cryostorage.…”

“…Despite these benefits, the adoption of neuronal cryopreservation methods has been limited ( Paynter, 2008 ). Pioneering studies found that neuronal cells could be frozen in “hibernation media” supplemented with cryoprotectant, which on revival yielded neurons that appeared morphologically and physiologically normal ( Kawamoto and Barrett, 1986 ; Mattson and Kater, 1988 ; Petite and Calvet, 1995 ; Otto et al, 2003 ; Quasthoff et al, 2014 ). However, despite optimization of freezing parameters and media composition, the viability and long-term survival of cryostored neurons was consistently low using these methods ( Higgins et al, 2011 ; Robert et al, 2016 ), rendering neuronal cryopreservation an inefficient practice.…”

Cryopreservation of Primary Mouse Neurons: The Benefit of Neurostore Cryoprotective Medium

“…Neurostore contains a proprietary combination of cryoprotective agents to reach a superior viability of primary cultures upon thawing. Other studies have proposed protocols to achieve a satisfactory cryopreservation of neural cells (Das et al, 1983 ; Fang and Zhang, 1992 ; Negishi et al, 2002 ; Higgins et al, 2011 ; Quasthoff et al, 2015 ; Robert et al, 2016 ). However, previously published studies measured mainly qualitative outcome of viability (Das et al, 1983 ; Fang and Zhang, 1992 ; Robert et al, 2016 ) or cell membrane integrity (Negishi et al, 2002 ).…”

“…Few authors characterized cryopreserved cultures in deeper detail. For example, Higgins et al ( 2011 ) provided a qualitative evaluation of neuritic tree formation and only Quasthoff et al ( 2015 ) included a parametric analysis of morphological and functional development of the cultures. In our study, we found no major differences in the viability of the fresh and frozen cells at different time points of their maturation.…”

Defining parameters of specificity for bioluminescent optogenetic activation of neurons using in vitro multi electrode arrays (MEA)

“…While there are membrane potential measurements that can only be carried out in single cells, assessment of neuronal populations can be highly informative as well. Multi‐electrode arrays (MEAs) allow recording of the electrical activity of neuronal networks grown on multiple extracellular electrodes (Berdondini et al, ; Chiappalone, Bove, Vato, Tedesco, & Martinoia, ; Illes, Theiss, Hartung, Siebler, & Dihné, ; Novellino et al, ; Otto, Go, Fleischer, & Siebler, ; Quasthoff et al, ; Schock et al, ). The advantages of this technology are its relative simplicity, compactness and ease of monitoring, as well as its potential for high throughput (Hales, Rolston, & Potter, ; Pine, ; Spira & Hai, ).…”