Neurons, cryopreservation

Since Harrison, in 1907, first microscopically observed neurite outgrowth from cultured frog neurons, many have experienced the powerful persuasion of direct visualization of cellular responses in culture. Standardization of media composition, introduction of antibiotics, and development of cryopreservation techniques and immortalized cell lines in the 1950s enabled routine application of cell culture in the biological research laboratory. Utilization of cell culture in toxicology soon followed with development of systems for monitoring chemically induced mammalian genotoxicity. 

The cryopreservation technique has been established for some cells7 As a result, these cryopreserved cells have been used in many studies. However, in the case of neurons, the appropriate cryopreservation process has not yet been established because of their complex structure, low mitotic activity, and uncertainties regarding the cryoprotection mechanism. Thus, primary culture cells are used for studying neurons. 

We studied the effect of cryoprotectants and cooling rates on the cryopreservation of rat cortical neurons. We then evaluated the recovering neurons by observing them by phase-contrast microscopy and by measuring their action potentials with the MED system. 

Primary rat cortical cells were prepared from neonatal Sprague-Dawley (SD) rats within 3 days of birth (Primary Cell Co., Ltd.). The dissociated cells contained neurons, glial cells, and fibroblasts. These cells were suspended in the culture medium at 1 X 10 cells/ml solution. The materials that were to be tested as cryoprotectants were then added to the medium. The culture medium consisted of Dulbecco s modified Eagle s medium (DMEM) and 10 vol% fetal bovine serum (FBS). Control dissociated neurons that had not been treated with the cryopreservation were cultured so that they could be compared with the cryopreserved neurons. 

Three types of cryoprotectants at various concentrations were selected DMSO (5-20 wt%), trehalose (3-17 wt%), and a mixture of trehalose (10 wt%) and methanol (8 wt%). A mixture of methanol and trehalose has been reported to be effective for the cryopreservation of salmon sperm.Therefore, this mixture was also used to check its effectiveness in the cryopreservation of neurons. 

Initially, we cultured the control neurons without cryopreservation. Neurons cultured on a poly-L-lysine-coated 24-well plate were examined by microscopy and immunofluorescence staining using MAP2 (Figures 1,2). Arrows indicate living neurons. 

Figure 1 Control cultured neurons that were not cryopreserved.

In order to check the effectiveness of the cryoprotectants, neurons were cryopreserved with regular culture medium (without cryoprotectants). Recovering cells were not observed in microscopic observations when cryopreservation was carried out using the same experimental protocol (Figure 4). In case of cryopreservation in the absence of cryoprotectants, the ice around the cells disrupts the cell membrane. 

Cryopreservation with 5-10 wt% DMSO showed that recovering neurons were present this was determined by microscopic observations and immunofluorescence staining using MAP2 (Figures 5,6). White arrows show living neurons, and black arrows indicate dead neurons. 

Figure 5 Recovering neurons (after cryopreservation with 10 wt% DMSO) obsei ed by phase contrast microscopy.

The number of neurons recovered after treatment with 10 wt% DMSO was reduced to approximately 60% of that obtained after treatment with 5 wt% DMSO. Cells were not present after cryopreservation with 20 wt% DMSO. These results indicate that neurons were successfully recovered after cryopreservation with low DMSO concentrations. Cryopreservation with 20 wt% DMSO appears to have a toxic effect on the cells. 

Furthermore, cryopreservation with 5 wt% DMSO preserves the nerve impulses of the recovering neurons as determined with the MED 64 system (Figure 7). 

As shown in the extended signal in Figure 3, the function of the neurons was assayed based on the presence of nerve impulses these nerve impulses were equivalent to those of neurons that had not been cryopreserved (amplitude 30 pV, width of pulse 3 ms). These results indicated that the DMSO-cryopreserved neurons (at DMSO concentrations of 5-10 wt%) can be recovered and cultured without any damage to their morphology or electrical activities. 

Figure 9 Cells stained with MAP2, a neuron-specific marker (after cryopreservation with 17 wt% trehalose).

Recovering neurons were not observed in the cryopreservation experiments with 3-10 wt% trehalose. Therefore, we consider that trehalose may only function as a cryoprotectant at concentrations of 17 wt% and possibly higher. In the cryopreservation experiments with 17 wt% trehalose, the recovery ratio of neurons was lower than that obtained with 5 wt% DMSO. The nerve impulses of the recovering neurons were not measured in this case because the recovery ratio of neurons was low. It has been reported that trehalose inhibits the growth of ice, and this property would prevent the disruption of the neuronal membrane. To determine if trehalose is a suitable cryoprotectant for neurons, further studies are necessary. 

Some neurons recovered after cryopreservation in 17 wt% trehalose at a cooling rate of 10 K/min as determined by microscopic observations and immunofluorescence staining using MAP2 however, neurons did not recover at lower concentrations of trehalose. These results indicated that trehalose functions as a cryoprotectant only at high concentrations. 

The difference in the recovery of cortical neurons in the presence of these additives might be a result of the cryopreservation process that is explained as follows DMSO is taken up into the cell and allows vitrification of the intracellular solution, leading to the stabilization of the cell membrane. However, in the case of trehalose, the molecules may be too large to permeate into the cell they may only contact the membrane extracellularly and protect the cell from freezing damage. To clarify the details of this cryoprotection mechanism, we need to study the process of cell freezing. 

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