The overall goal of this procedure is to isolate human neural stem cells from discarded frontal cortical tissue. This is accomplished by first preparing the solutions and materials in advance before dissecting human fetal brains. This is followed by the maintenance of isolated neural stem cells, including further expansion, characterization, and experimentation.
Long-term storage is feasible as is establishment of subcultures for differentiation of progenitors into neuronal and glial cell types. Ultimately, the differentiation of cells from normal CNS can be characterized and compared to that of cells derived from CNS with known neurological disorders. The implications of this technique extend towards therapy or diagnosis of any human CNS disorder.
The technique provides a simple and reliable platform to evaluate pathological S and to assess pharmacological efficacy of new drugs for given disorder. Because of the exceptional value of multipotent cells, all materials need to be at the ready in advance of starting the protocol. To minimize risk of compromising the samples have prepared refrigerated dissection, medium bath warmed 37 degrees Celsius culture, medium and refrigerated cell, freezing medium for long-term.
Cryo-preservation for the dissection, have sterile autoclaved forceps and scalpel blades with handles ready for cell dissociation. Have a pipette gun, 10 milliliter transfer pipettes, and a 40 micromolar cell strainer. Ready, set aside several 10 centimeter culture dishes for dissection.
50 milliliter centrifuge tubes for dissociation. 1.5 milliliter centrifuge tubes for tissue storage and frozen vials for freezing cells To ensure success. 18 to 22 week old viable tissue needs to be harvested within two hours of its availability.
Ideally, fetal tissue can be had within minutes of its availability by making arrangements to collect after elective procedures. The tissues often are fragmented, however, a significant portion of the brain remains intact. Collect the brain in a 10 milliliter Petri dish filled with ice cold dissection.Medium.
Identify different parts of the cortex by anatomical landmarks. Boundaries for the frontal and parietal occipital cortices are oriented by the extrapolated intersection of the central sulcus and syl fissure. Dissect tissue from the frontal cortex anterior to the central sulcus and along the border of the sylvan fissure with surgical blades.
Make sure to keep the ventricle intact and undamaged. Now, remove any residual blood and meninges from the separated block of frontal cortex. If the sample is not too fragmented and is sufficiently large, dissect the block into several smaller samples to save some for histology and protein or mRNA assays.
Now transfer a brain block to a 50 milliliter centrifuge tube and add three times the tissue's volume of ice cold dissection medium. Using a pipette, gently dissociate the tissue until all the tissues become fragmented. To fully suspend the cells, filter them through a 40 micromolar cell strainer.
Now centrifuge the cell suspension and resuspend the cell pellet in 10 milliliters of fresh, warm culture medium. Using a hemo cytometer, count the cell number and calculate the volume for 2 million cells. Now transfer each 2 million cell volume to a 25 centimeter square culture flask.
Prepared with five milliliters of warm culture medium. Maintain the cells at 37 degrees Celsius with 5%carbon dioxide for one week. Change half the medium once a week for longer cultures After one to two weeks in culture, neurospheres usually form with an NSC diameter between 200 and 400 microns.
To dissociate the neurospheres into single cells. Incubate the cells with EDTA in Hank's medium for 15 minutes. To prepare the suspended cells for subculture, spin them down, wash with hanks and repl the cells in warm culture.Medium.
To initiate differentiation, plate the dissociated cells on poly de lysine. Laminin one coated cover slips at a density of 100, 000 cells per cover, slip and incubate for 30 minutes. The cells can easily be differentiated into oligodendrocytes neurons or astrocytes.
For neuronal differentiation, maintain the cells in medium with B 27 for seven days. For astrocyte differentiation culture, the cells in medium with FBS for seven days. For oligodendrocyte differentiation, maintain the cells in modified medium with growth factors for two days and after two days, use the same modified medium without the growth factors for another five days to store the stem cells.
After the dissociated cell suspensions are centrifuged, resus, suspend the cells in freezing medium at a concentration of 10 million cells per vial. Per milliliter, slowly freeze the cells at minus 20 degrees Celsius before freezing them at minus 80 degrees Celsius for long-term storage. Transfer them to liquid nitrogen.
Later the cells are fast thawed with a 37 degrees Celsius water bath and resuspended. In warmed D-M-E-M-F 12 plus 10%serum for a wash. Warmed cells are then centrifuged, aspirated, and resuspended in the warmed culture.
Medium neural stem cells from a normal fetus. At 18 weeks gestational age, were cultured to form neurospheres, which were transfected with GFP. The neurospheres were differentiated to make several cell types that were then fixed and stained with cell markers and rumine.
Multi potentiality is observed with expression of markers, indicative of neurons, astrocytes, and oligodendrocytes. After watching this video, you should have a good understanding of how to isolate, expand, and manipulate human neuro stem cells from discarded frontal cortical tissue. This regions provide a unique opportunity to study human cortical environment disease maximums, and test the potential efficacy of pharmacotherapeutic agents.
