Gibco Hibernate Media

Figure 1. Live/Dead cell viability assay showing live cells stained with Calcein-AM (green) and dead cells with EthD-1 (red). The viability of primary rat hippocampal and cortical neurons maintained in HIBERNATE-E Medium at ambient conditions was equivalent to the viability of neurons in Neurobasal Medium in a standard 5% CO₂ incubator. Corresponding cultures of neurons maintained in Neurobasal Medium at ambient CO₂ conditions showed dramatic loss of viability at 48 hours.

Figure 2. The viability of embryonic rat cortical neurons in Hibernate-E Medium, and post natal (P1) cortical neurons was maintained in Hibernate-A Medium at ambient conditions after 4 days in standard culture conditions. Cells maintained in Hibernate media at ambient CO₂ showed less than 10% viability loss as compared to the control i.e., neurons in Neurobasal media in a 5% CO₂ incubator. Neurons maintained in complete Neurobasal media at ambient CO₂ conditions showed more than 70% viability loss by 48 hours. Calculated means and standard errors were tested for significance by ANOVA (n=3, p<0.001).

Figure 3. Glutamate-induced changes in intracellular calcium levels were measured with the Fluo-4 Direct Calcium Assay Kit. Pseudocolored images before (top) and after (bottom) challenge with 500 μM glutamate are shown. Purple represents low and red represents high intracellular calcium. The bar graph indicates that most neurons in Neurobasal Medium at 5% CO₂ responded to glutamate (116/119), few neurons in Neurobasal Medium kept at ambient CO₂ responded to glutamate (5/13) and most of the neurons kept at ambient CO₂ in Hibernate-E Medium responded to glutamate (98/100).

Figure 4. Growth of HUVECs from tissue shipped in Hibernate medium or DMEM. Freshly isolated human umbilical cords were cut in half. One half was shipped overnight in wet ice in Hibernate-E medium, while the other half was shipped in DMEM under the same conditions. Cords were unpacked, examined for any damage and then both halves were trimmed to equal lengths. HUVECs were isolated and cultured from each half by standard protocols using the same reagent, medium, and supplement.  Cultures were grown until 80% confluent, then both sets were harvested and the cells were counted. The graph demonstrates that the growth of HUVEC from cords shipped in Hibernate-E medium is significantly better than growth of cells from cords shipped in DMEM.