High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons

Abstract

Cell cryopreservation improves reproducibility and enables flexibility in experimental design. Although conventional freezing methodologies have been used to preserve primary neurons, poor cell viability and reduced survival severely limited their utility. We screened several high-performance freezing media and found that CryoStor10 (CS10) provided superior cryoprotection to primary mouse embryonic cortical neurons compared to other commercially-available or traditional reagents, permitting the recovery of 68.8% of cells relative to a fresh dissection. We characterized developmental, morphometric, and functional indicators of neuron maturation and found that, without exception, neurons recovered from cryostorage in CS10 media faithfully recapitulate in vitro neurodevelopment in-step with neurons obtained by fresh dissection. Our method establishes cryopreserved neurons as a reliable, efficient, and equivalent model to fresh neuron cultures.

Aberrant activation of Rho/ROCK signaling in impaired polarity switching of colorectal micropapillary carcinoma

“…pPB/RhoA (G14V) and pPB/RhoA (WT) were generated by transferring the fragments of the RhoA (G14V) and RhoA (WT) genes, respectively, to the pPB Puro vector. pLenti Lifeact‐mRuby2 BlastR was a gift from Ghassan Mouneimne (Addgene plasmid #84384) [15] and transfected as follows. For the generation of lentiviral particles, pLenti Lifeact‐mRuby2 BlastR was transfected to HEK293FT cells with lentiviral packaging constructs pMD2.G and psPAX2 (Addgene #12259 and #12260, both gifts from Didier Trono) using X‐tremeGENE HP transfection reagent (Roche).…”

Neuronal Cell-based Medicines from Pluripotent Stem Cells: Development, Production, and Preclinical Assessment

“… 45 Recently, some studies have selected suitable cryopreservation solutions for neuronal cells. 46 , 47 These require appropriate cooling rates and thawing conditions validated for each neuronal cell product, suggesting that precisely controlled cooling and warming conditions will further improve cell viability. 48 Notably, the same study showed that the viability and recovery of cells at 24 hours post‐thawing could more accurately reflect the effect of cryopreservation and this is a principle now well established in cell cryobiology.…”

EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in developing neurons

“…Following three five-minute rinses in HBSS, digested tissue was triturated 20 times each with three P1000 pipette tips cut to progressively smaller gauges. The homogenate was passed through a 70µm filter, counted, and cryostored as described (Parker et al, 2018). Isolated cells were resuspended in CryoStor CS10 (BioLife Solutions, 210102) to 6 million cells per mL, aliquoted, and cryostored at -80°C for at least two days then transferred to liquid nitrogen.…”

“…Validated Mus musculus shRNA sequences were obtained from The RNAi Consortium (The Broad Institute via MilliporeSigma, (Moffat et al, 2006)) and oligos were ligated between the AgeI-EcoRI sites (replacing the 1.9kb stuffer) of our pLKO.1 TurboRFP cloning vector using annealed oligo cloning. shRNA sequences and source MilliporeSigma product number are given in Key Resources Table . Lentiviral LifeAct expression vectors were published previously (Padilla-Rodriguez et al, 2018;Parker et al, 2018): pLenti LifeAct-EGFP BlastR (Addgene #84383; RRID:Addgene_84383), pLenti-LifeAct-mRuby2 BlastR (Addgene #84384; RRID:Addgene_84384), pLenti LifeAct-iRFP670 BlastR (Addgene #84385; RRID:Addgene_84385). Lentiviral cDNA expression vectors were generated by PCR and subcloning cDNA of interest into the transfer plasmids pLenti CMVie-IRES-BlastR or pLenti CMVie-IRES-BlastR alt MCS (pCIB) published previously (Puleo et al, 2019) (Addgene #119863 and #120862; RRID:Addgene_119863 and RRID:Addgene_120862).…”