Análise de Desenvolvimento de dente Germ Innervation Utilizar dispositivos microfluídicos Co-cultura

The overall goal of this procedure is to show how to isolate and co-culture the developing trigeminal ganglia and tooth germs. This is accomplished by first sterilizing, mounting and coating the microfluidic devices. The second step is to dissect trigeminal ganglia and tooth germs from a specific developmental stage in the mouse of interest.

Next, the trigeminal ganglia and the tooth germs are placed into the microfluidic devices and co cultured. The final step is to fix the cells and stain them using immunofluorescence. Ultimately, microscopy is used to show the innervation pattern that is the result of co-culture.

The main advantage of this technique of existing methods, such as conventional co cultures, is that this method allows the co culture of different organs and tissues each in their own optimal culture.Medium. This method can help answer key questions in the field of artificial innovation, such as the role of different molecules on tooth innovation and the rule of innovation in tooth development. Begin by autoclaving dissection tools, which include a pair of microdissection forceps and a pair of scissors.

Next, sterilize a batch of 24 millimeter by 24 millimeter glass cover slips by placing them into a solution of one molar hydrochloric acid on an orbital shaker at 37 degrees Celsius for 24 hours. Then wash the cover slips three times with sterile distilled water, followed by three rinses in 99%Ethanol, dry them under a sterile flow hood once dry, turn on the hood UV light for 30 minutes. Then store the cover slips in 70%ethanol until they are needed.

Next, use sterile forceps to carefully remove the axon isolation microfluidic devices from their packaging and place them into a sterile Petri dish using a sterile one millimeter biopsy punch. Create one hole in the devices for each sample. In correspondence of the culture chambers.

Be careful not to punch too close to the micro grooves as they might be damaged by the pressure. Next, sterilize the devices by emerging them in 70%ethanol. Taking care to remove all of the trapped air.

Then remove the devices from the ethanol and dry them completely under a sterile flow hood. Also, remove the cover slips from the 70%ethanol solution and let them dry under the sterile flow hood. Wait a minimum of three hours before proceeding.

Place each cover slip into a well within a six well plate. Then place the microfluidic device onto the cover slip and press gently but firmly with the forceps in order to allow full adhesion between the isolation device and the glass cover slip. Next, pipette 150 microliters of 0.1 milligrams per milliliter, poly D lycine into each of the culture chambers.

Then place the microfluidic devices under vacuum for five minutes to remove all the air from the chambers. If air can still be seen within the chambers. Repet the poly D lysine solution into the chambers.

Incubate the devices with poly de lysine overnight at 37 degrees Celsius the next day. Wash the chambers three times with sterile distilled water, and then fill the chambers with 150 microliters of five micrograms per milliliter, lamin working solution, and incubate them overnight at 37 degrees Celsius. Next, repair media for the trigeminal ganglia and tooth germ cultures as described in the accompanying text protocol.

Clean the dissection area and the stereoscope with ethanol 70%and place the dissection instruments following sacrifice. Dissect the skin around the lower abdomen of a pregnant mouse with stage E 14.5 to E 17.5 mouse embryos. Then carefully open the abdomen using scissors.

Locate the uterus, remove it, and place it into a tube filled with ice. Cold PBS dissect out the embryos and place them individually into a new Petri dish filled with ice cold PBS. Once all the embryos have been removed from the extra embryonic tissues, decapitate them using scissors.

Separate the lower jaw from the rest of the head using micro dissection, scissors, taking care not to damage the trigeminal ganglia. Next, take the head and place it onto a chilled dissection glass Petri dish. Use forceps to remove the skin and the skull.

Then place forceps below the cephalon and lift. To remove the cephalon and the cerebellum, localize the trigeminal ganglia and use the forceps and dissection needles to separate the trigeminal ganglia from the trigeminal nerves and clean the ganglia. Preserve them in cold PBS using dissection needles as knives.

Remove the tongue and the skin surrounding the jaw. Separate the left and the right hemi jaws by cutting along the midline of the jaw. Then locate the tooth germs and isolate them using dissection needles after dissection of the trigeminal ganglia and tooth germs, remove the laminin from the microfluidic devices and fill the chambers with 200 microliters of the respective media.

With forceps, gently transfer the dissected trigeminal ganglia and tooth germs into the holes created with a biopsy punch. Make sure that the tooth germs do not float and that they sink until they contact the cover slips. Culture, the samples in an incubator at 37 degrees Celsius and 5%carbon dioxide.

Change the culture medium every 48 hours for 10 days to change the medium. First, remove the medium by pointing the pipette towards the external side of the wells. Then add the fresh prewarm medium on the side of the wels located opposite to the chambers during the culture period.

Image the co cultures at any time using light microscopy. After the culture period, wash the chambers by pipetting 150 microliters of PBS into one well per chamber, and letting PBS flow through the chambers. Repeat the wash a total of three times following the last wash.

Remove the PBS and fix the samples by pipetting 150 microliters of 4%paraform aldehyde in PBS into one well per chamber, and allowing it to flow through to the other chamber. Incubate the device at room temperature for 15 minutes. Then wash the chambers twice with PBS and proceed with further analysis such as immunofluorescent staining and imaging of the outgrowth.

During coal culture, neurites from the trigeminal ganglia branch out from its culture compartment and extend towards the developing tooth germ. At a higher magnification, one can see the neurites extending through the axonal chambers and the micro grooves in the microfluidic device. The progress of nerve growth can be tracked over time to describe the innovation development.

Following this procedure, other methods like RNA or PROTEINIZATION can be performed in order to study, for example, changes of gene expression or protein secretion in the target tissues following innovation.