The overall goal of this procedure is to isolate nasal olfactory stem cells from rats or humans in order to identify molecular anomalies or repair the pathological or traumatized brain. This is accomplished by first surgically collecting olfactory mucosa biopsies from the nasal cavity. Next, eliminate the olfactory epithelium and its epithelial stem cells to focus on ecto mesenchymal stem cells in the underlying Lamin propria, and then isolate those cells by dissociating rodent tissue, or collecting outgrowing cells from human implants cultivated under glass cover slips.
The final step of the procedure is to grow stem cell spheres inducing differentiation into neuron like cells. Ultimately explan of the whole olfactory mucosa, individualized stem cells. Floating spheres or neuron like cells can be used for identifying molecular markers in brain diseases or repairing the damage to nervous system.
We describe her method for isolating human and rodent nasal stem cells from the olfactory mucosa. The main advantage of this technique over existing method is that the stem cells belong to a nervous tissue, are easily accessible in every living individual, and can be used for diagnosing or repairing the pathological brain. As an ENT surgeon, I've performed hundreds of nasal biopsies for fun clinical studies.
The procedure is safe and it can be carried out. And the local general anesthesia. I'm a junior scientist with an expertise on the culture of human or rod, nasal or factor stem cells.
This has display hyper preparation rate and an inclination to differentiate into neuro cell types. They're proven to be efficient in animal models of amnesia and backing sense disease In advance of collecting the mucosa. Biopsies have 3 35 millimeter Petri dishes with DMEM Ham, F 12 culture, medium readied in a clean culture hood.
Now after verifying via toe pinch that the ratt has entered deep anesthesia with sodium pentobarbital euthanize by decapitation. Isolate the head and remove the skin. Remove the lower jaw with scissors and with the help of her own juror, eliminate the facial muscles on both sides starting from the back of the incisors.
Using the Ron Jur. Remove the bone covering the nasal cavity one side at a time. The olfactory turbinates come into sight as orange, brown organs located in the back of the nose, delicately discard the turbinates with forceps.
Using a 26 gauge needle, isolate the olfactory mucosa lying on the septum by cutting the tissue along three lines, the arc of the perpendicular plate, the CRI form plate, and the ceiling of the nasal cavity. Collect biopsies on both sides and transfer them to A-D-M-E-M ham F 12 filled Petri dish. This procedure should take no longer than 10 minutes from the onset of euthanasia.
Now in order to remove the mucus, transfer the biopsies two times in medium filled Petri dishes In accordance with the relevant local ethical committees. An ear, nose, and throat surgeon should carry out this procedure and every outpatient should sign an informed consent form using a zero degree or 30 degree rigid endoscope. Inspect both nasal cavities and assess the putative presence of polyps or any inflammatory lesion.
Choose the best nasal cavity taking into account the deviation of the septum. Using a cotton applicator, apply a local anesthetic such as lidocaine with epinephrine for 10 minutes with a through cut. Ethmoid forceps collect a two square millimeter biopsy either at the root of the medial aspect of the middle turbinate or on the septum in the dorsal medial area.
Here we show the biopsy from the root area. The olfactory biopsy is then transferred using a sterile needle into a sterile two milliliter tube filled with one milliliter of DMEM hem F 12. Tip the tube upside down to make sure that the biopsy is immersed in the culture medium.
Insert the tube in a refrigerated container and transport it to the research laboratory. At this stage, the biopsy can be used per se for comparative molecular studies focused on specific brain diseases or processed for generating stem cells. Wash the human or rat biopsies and DMEM ham F 12 incubate the biopsies in a Petri dish filled with one milliliter of dis paste.
Two solution for one hour at 37 degrees celsius. Next, the human or rat olfactory epithelium is removed from the underlying Lamin propria using a micros spatula under a dissecting microscope with a diffracted inverted light. The olfactory epithelium is translucent over a black background compared to the lemon propria, which is striped orange brown over a white background.
The epithelium looks gray and the Lamin propria brown once purified, transfer the lemon propria into a Petri dish filled with DMEM hem F 12. If the tissue is from a rodent, cut the lemon propria into small pieces with 2 25 gauge needles. Then transfer the pieces to a 15 milliliter tube filled with one milliliter of collagenase one A in the tube, using a sterile plastic pipette to dissociate the tissue.
Then incubate the tube for 10 minutes at 37 degrees Celsius. To terminate the dissociation, gently rock the tube and add milliliters of calcium free and magnesium free PBS and centrifuge at 200 Gs for five minutes. Resus, suspend the cell pellet in culture medium and play on plastic culture dishes.
Now, if the tissue is human, slice the lemon appropriate into three to four pieces. Insert each strip into its own two centimeter diameter. Well in a four well plate and cover the tissue with sterile 1.3 centimeter diameter glass cover slips.
Then add 500 microliters of culture medium to each culture dish for either tissue type. Renew the culture medium every two to three days. After five to seven days of culturing, the stem cells will begin to invade the culture dish.
And after two weeks, they should be confluent. When co fluency is reached. Passage and transfer the cells to culture flasks to generate stem cells spheres.
Replay the cells at a density of 16, 000 cells per square centimeter into pretreated flasks every two days. Feed the cells with 0.2 milliliters per square centimeter of supplemented medium. Two to five days later, collect floating cell spheres and either repl or dissociate them before grafting.
An animal model of cell therapy to differentiate the olfactory stem cells into a neuron like cells. Cultivate the cells for 21 days in neuro basal medium containing B 27 penicillin, streptomycin, glutamine, and glutamate then make medium changes every three days. Neuron like cells should appear after two to three weeks, five to seven days post plating.
Nasal human olfactory stem cells grew out of the explan and proliferated rapidly. Conf fluency could be reached within a week or two. A key feature of stem cells, nest and expression was evaluated when grown on polylysine with a serum free culture medium supplemented with EGF and FGF two, the olfactory stem cells gave rise to spheres when the spheres were plated in serum containing culture.
Medium, they gave rise to cell populations containing approximately 50%GFAP expressing cells up to 15%tubulin expressing cells, and up to 5%oh four expressing cells. When the sphere derived cells were grown in supplemented neuro basal culture medium, most of the population became neuron like cells expressing beta three tubulin. The same neuron like cells also expressed map two.
Thus, the fate of the stem cells could be modified One semester. This technique can easily generate and rest of million stem cells in less than four weeks, allowing the clinicians to perform future autologous cell transplantation. Nasal refractory biopsies are very useful for identifying biomarkers and brain diseases.
Recently we have been successful in finding new candidate genes for autism. We have already set Up a back of olfactory stem cells from autistic and controlled individuals, and it can be enlarged to other human brain disorders.
