Hello, my name is Alan and I'm a Dr.Horney Lu's lab at the University of California Irvine in the Department of Neurobiology and Behavior. And I'm Bob. Today, Dr.Haro will be showing you a fast, efficient, and reliable protocol for the routine cryo preservation of cortical tissue blocks for the subsequent degeneration of high quality primary neuronal cultures.
So let's get started. Cleaned and chopped tissue is loaded into cryo vials containing a 10%TMSO. Freezing medium.
Cryo vials are loaded into a freezing container and placed at negative 80 degrees Celsius for at least four hours. Frozen cryo vials are then placed in liquid nitrogen for long-term storage. For culturing AC cryo vial is melted in a warm water bath and rinsed with warm HBSS to remove the remaining DMSO.
Fresh HBSS is added along with tryin d ns for dissociation, after which DMM with 10%EBS is added. To stop dissociative enzymatic activity cells are spun down and the pellet resuspended in fresh DMM plus 10%EBS and plated on pre-coated polylysine dishes. After 24 hours, DMM has changed to a supplemented neuro basal medium and grown for 10 days.
This protocol uses a steel razor blade for chopping cortical tissue before use. The razor must be sterilized in 70%ethanol for at least two hours. One x Hank's buffered salt solution HBSS is created by diluting a 10 x stock solution in sterilized water, the one X-H-B-S-S is then either kept on ice or at four degrees Celsius until tissue processing.
Dimethyl sulf oxide or DMSO is used as a cryo protected in the freezing media. This protocol uses a 10%volume and volume dilution of DMSO in one X-H-B-S-S-D-M-S-O should mix completely before storing at four degrees Celsius. Nalgene and freezing container is used to control the rate of freezing for the samples.
The freezing container is loaded with 2.5 milliliter cryo vials that have been labeled beforehand. It's also a good idea to label the freezing containers themselves if you have more than one and note the corresponding container on the bile. The previously chill 10%DMSO, freezing media, as well as the freezing container are brought into a sterile biological safety cabinet.
Here one milliliter of freezing media is slowly added to each of the cryo vials After making sure the caps are secure, place the freezing container with the vials at four degrees Celsius for at least two hours just prior to tissue processing. The previously sterilized razor blade needs to be rinsed thoroughly. Three, three minute washes.
Using sterilized water should be sufficient for removing any remaining ethanol. This washing procedure should be quick as the razor is prone to oxidation from prolonged submersion and sterile water. Before cleaning tissue should be placed on an ice pack.
Rinse slightly with cold one X-H-B-S-S to remove any free floating debris. Sterilized needles are used to clean the tissue pieces of any remaining membranes and blood vessels. Care must be taken to preserve structural integrity of the tissue while cleaning.
As this will increase, the efficiency of chopping clean tissue will be visibly different than raw tissue and should have most of the membranes or blood vessels removed. Once a tissue has been cleaned and rinsed, chopping can begin. It's important to make clean cuts of the tissue creating blocks approximately one millimeter cubed in size.
Blocks that are too big level a lower cell yield at thawing. The smaller blocks, however, overprocessing should also be avoided. Chopped tissue can be collected using an auto pipette and some cool one X-H-B-S-S.
Make sure to rinse the dish with HBSS to recover all tissue collected. Tissue is then added to a large volume of one X-H-B-S-S and allowed to fall to the bottom, creating a loosely packed pellet. This cleans out any remaining debris that may be present as the tissue is settling in the HBSS retrieve the previously chilled freezing container and cryo vials before allocating tissue.
Make sure to uncap all the vials to hasten the process of filling tissue. Exposure to DMSO at room temperature should be limited to no more than 10 minutes. It's a good idea to work with one freezing container at a time until all tissue is used.
Begin to aspirate the supernate of a loose tissue, pellet corn as close to the pellet as possible without disturbing it. Then fit a 200 microliter wide orifice tip to a pipette. Do not use a smaller tip orifice as it will compromise the structure of the blocks going to the bottom of the tissue block pellet.
Collect 200 microliters of tissue slow. Add this tissue to a cryo vial and move on to the next until all vials are filled. Might need a 10 minute rule.
Quickly place the freezing container in cryo vials a negative 80 degrees Celsius freezer for at least four hours or overnight if the time is limited. After sufficient time, remove the freezing container from the freezer and allocate the cryo vials to a cryo box as quickly as possible. Place the cryo box in a liquid nitrogen box rack for long-term storage.
Remove a cryo vile from liquid nitrogen as quickly as possible, limiting the cryo boxes Exposure to room temperature rapidly melt the sample by submerging in a 37 degree Celsius water bath. Always keeping the cap above water. Check frequently and keep thaw until a small ice P is observed.
Before bringing it in the safety cabinet, sterilize the cryo vile with 70%ethanol. Proceed to add the thaw tissue to a large volume of warm one X-H-B-S-S if tissue remains on the walls of the cryo vile. Gently collected with HBSS.
Once all tissue was collected. Invert the tube three to four times and allow the tissue to settle to the bottom. This step ensures sufficient dilution of DMSO.
Once the tissue is settled, aspirate as much of the super natin as possible. Then add 10 milliliters of warm fresh one X-H-B-S-S to this. Add 300 microliters of 0.25%trypsin and 50 microliters of DNAs.
Proceed to dissociate the tissue with gentle agitation, creating a cloudy cell suspension after the tissue is dissociated. Stop the enzymatic activity by adding warm DM a plus 10%enriched bovine serum EPS centrifusion cell suspensions at 1200 G for five minutes. Resuspend the cell pellet in fresh DMEM plus 10%EBS and plate to polylysine coated plates.
Allow cells to attach to dishes for 30 minutes. Then replace the media with fresh DM plus 10%EBS. After 24 hours, replace DM contained media with neuro basal media supplemented with N two and B 27 and grow for 10 days.
We have just detailed a vast, efficient, and reliable protocol with a routine cry preservation of cortical tissue blocks. So good luck.
