Organotypic cultures of human adult cortex are so far the only system for testing stem cell therapies for damaged cortex that allows studying the interaction between human grafted and human host cells. A therapy tested in animal models usually fail when translated to the clinical settings due to the differences between rodent and human cells. Here, we can study grafted cells, survival, differentiation, and functionality in a human-to-human setting.
This methodology will help us to understand how the neural circuitry operates in human brain, and also it'll stimulate the translation of this knowledge into the clinical settings to improve the functional recovery after brain damage. Demonstrating the procedure will be Raquel Martinez-Curiel, a PhD student, and Costanza Aretio-Medina, a masters student, both from my lab. To begin, place the culture inserts in a six-well plate using forceps in the cell culture lab under a ventilated hood.
Add five milliliters of the human adult cortical medium on the bottom of the insert until it contacts the membrane. Avoid bubble formation and add two milliliters of the medium on top of the insert. Before transferring the tissue slices into the inserts, equilibrate them in the incubator at 37 degrees Celsius and 5%carbon dioxide for at least two hours.
Collect the tissue from the patient in the operating room into a container of frozen, bubbled, and crushed cutting solution. Transfer the closed container on ice immediately to the cutting area of the lab. Inspect the tissue, and considering the cortical layer's orientation, locate the best surface to glue it to the vibratome's cutting stage.
If needed, cut the uneven surface with the scalpel to place the tissue on the stage for optimal slice orientation. Then, glue the tissue to the stage with tissue adhesive. Place it in the slicing chamber and immediately fill it with cold, bubbled cutting solution.
Continue the bubbling during the whole cutting procedure. Now cut coronal or sagittal slices depending on the tissue-to-blade orientation as described in the manuscript. Place the slices in the collection chamber with bubbling cutting solution at room temperature.
Once all the tissue has been cut, transfer the slices into a sterile Petri dish with rinsing solution at room temperature to remove excess sucrose from the slices and transport them to the cell culture lab. Next, place the tissue slices individually on top of the wet and submerged inserts. After 24 hours, change the medium to remove any remaining sucrose or other residual substances from the cutting procedure.
After two weeks, use human adult cortical medium without gentamycin. Remove the media from the cell culture flask. On day seven of differentiation, detach cortically-primed GFP-lt-NES cells by adding 500 microliters of trypsin and incubate for 5 to 10 minutes at room temperature.
Next, add an equal volume of the trypsin inhibitor followed by five milliliters of DDM medium. Resuspend the cells, gently transfer the cell suspension into a 15-milliliter tube, and centrifuge for five minutes at 300 G.Meanwhile, transfer the plate containing the human tissue from the incubator to the hood, and remove two milliliters of media from the top of the insert. At the end of the centrifugation, remove the supernatant, resuspend the cells in a cold, pure, basement membrane matrix, and transfer the pension to a smaller tube.
Collect the cell suspension into a cold glass capillary connected to a rubber teat for suction. Inject the cell suspension as tiny drops by stabbing the semi-dry tissue slice at various sites. Allow the gel to solidify at 37 degrees Celsius for 30 minutes.
Then, transfer the plate from the incubator back to the hood and carefully add two milliliters of human adult cortical medium to the top of the insert to completely submerge the tissue. The acute human adult cortical tissue showed the presence of neurons, oligodendrocytes, and astrocytes displaying optimal preservation of the tissue. The tissue showed the expression of neuronal markers NeuN and Map2 after two weeks of cell culture.
The whole cell patch clamp recording showed that neurons had sustained resting membrane potential and membrane input resistance, comparable to neurons from acute preparations. The cells were slightly less active than in fresh tissue, although most of the cells were able to fire at least one if not multiple action potentials. The acute cortical tissue showed the expression of microglial markers Iba1 and Tmem119.
After two weeks in culture, the microglia became less ramified and acquired a more activated morphology than in acute tissue. Four weeks after ex vivo transplantation, the grafted GFP-lt-NES cells exhibited extended neurites with extensive and complex arborizations throughout the whole organotypic culture. Barely any grafted cells survived in poorly preserved tissue.
Debris and unspecific labeling of antibodies on dead cells were broadly observed throughout the human slice. In the case of successful transplantation, the cells became functionally active and mature neurons showed repetitive and often spontaneous action potentials. Fast inward sodium, slow outward potassium currents, and a certain level of synaptic activity indicate functional integration of the graft with the host tissue four weeks post-grafting.
The faster the human tissue is processed once it is out of the operation room, the higher the viability of the system and the success of the experimental procedure.
