The overall goal of this procedure is to speed up the analysis of neuronal differentiation of human neural progenitor cells cultured in a three dimensional environment. This is accomplished by first culturing the cells in the self assembling peptide based hydrogel pure matrix. Next, the cells are released from the three dimensional scaffolds.
In the third step, immuno phytochemical staining of the cells is performed in the final step. The cells are enumerated by flow cytometric analysis. Ultimately, the rate of neuronal differentiation or the rate of apoptosis can be determined through the flow cytometric analysis for neuronal or apoptotic markers respectively.
With this technique, the quantification of neural differentiation is much faster and the Optane data west up in a wider base and micro photograph techniques For the embedding of cells in the pure matrix first place a sterilized 13 millimeter diameter glass cover slip into each of two wells of a four well cell culture plate. Then store the plate on a clean bench for later use. Next, detach the human neural progenitor cells or H NPCs by adding 2.5 milliliters of freshly prepared trypsin.
Benzon a solution to the culture dish and incubating the cell suspension at 37 degrees Celsius and 5%carbon dioxide. After five minutes, stop the reaction with trypsin inhibitor benzon a solution. Then after spinning down the detached cells at 3000 times G at room temperature for five minutes, resus suspend the resulting pellet with five milliliters of a 10%sucrose solution, and then wash the cell suspension under the same centrifuge conditions.
After discarding the snat Resus, suspend the pellet in the sucrose solution to a final cell concentration of one times 10 to the sixth cells per milliliter. Now as quickly as possible, mix 60 microliters of cell suspension with solution one transfer 120 microliters of this suspension to the conical tube with solution two and mix carefully and then immediately layer 100 microliters of the mixture onto each of the cover slips in the four well plate. Finally slowly add 200 microliters of cell culture medium to each well.
After two to three minutes, add another 200 microliters of cell culture media to the wells initiating the self-assembly of the matrix. Incubate the cells at 37 degrees Celsius for one hour on the heated area of your clean bench. Refraining from moving the cell culture plate as much as possible after the matrices have assembled, use a 1000 microliter pipette to remove most but not all of the media to avoid the matrices drying out.
Then wash the matrices with 500 microliters of fresh media for 10 minutes. Finally, remove the wash media. Add 500 more microliters of media and place the culture plate in an incubator.
If possible. Store the matrices in their own incubator to avoid accidental shaking of the structures on the first day of the staining experiment. Remove the media from the matrices with a 1000 microliter pipette and then wash the scaffolds with PBS once for five minutes.
Then remove the PBS and add 400 microliters of paraldehyde to each well to fix the matrices and incubate them for 30 minutes at room temperature. After the scaffolds have been fixed, wash them with 500 microliters of PBS for five minutes. Then incubate the structures and blocking buffer for six to nine hours, changing the buffer once every two to three hours with a final overnight incubation in fresh blocking buffer at four degrees Celsius the next day, remove the blocking buffer.
Add the antibody solution to the wells and incubate the matrices again at four degrees Celsius overnight. Now, remove the primary antibody solution and wash the scaffolds in 500 microliters of PBS for eight hours, changing the PBS every two hours and then subsequently overnight at four degrees Celsius with fresh PBS. After the overnight wash, remove the PBS and incubate the scaffolds with the secondary antibody solution for four hours at room temperature in the dark.
Then wash the samples with PBS four to six times for one hour each time, and then incubate the structures with fresh PBS at four degrees Celsius in the dark overnight. Now add 50 microliters of MOE all dab co onto the microscope slides to prepare them for mounting of the matrices. Then carefully mount a cover slip onto each slide.
Finally, use a fluorescent microscope to obtain single micrographs and Zacks similar to the H NPCs staining green for the expression of the neuronal marker beta three tubulin shown here. After washing the scaffolds with 500 microliters of HBSS for one minute, use a 1000 microliter pipette to transfer the scaffolds to a 15 milliliter conical tube. Next, resuspend the cell solution several times with a 1000 microliter pipette to mechanically disrupt the matrices and subsequently centrifuge the scaffold solution at 3000 times G at room temperature for five minutes, remove the supernatant with a pipette and then resuspend the pellet in 500 microliters of trypsin benzine solution by pipetting up and down several times.
Then incubate the cells at 37 degrees Celsius. Stop the reaction after five minutes with one milliliter of trypsin inhibitor. Benza nase solution pipetting up and down several times after spinning down the cell suspension under the same centrifuge conditions.
Remove any matrix debris by washing the cells with two milliliters of HBSS buffer two times. Finally, remove aggregates by passing the cell suspension solution through a 70 micron cell strainer centrifuge under the same conditions and collect the cells in cell culture medium. Begin by fixing the disaggregated cells in 1%paraform paraldehyde for 15 minutes at room temperature on a shaker.
Then after spinning down the cells reus, suspend the pellet in wash buffer. Now after centrifuging the cells for 10 minutes at 350 times G at four degrees Celsius reus suspend the resulting pellet in 25 microliters of the antibody solution. Incubate the cell suspension for two hours at room temperature on a shaker.
Then add 300 microliters of pon buffer directly to the samples, and then wash the cell suspensions twice under the same centrifuge conditions. After the second wash, add 25 microliters of the secondary antibody and then incubate the cells for one hour at room temperature in the dark. Then after washing the cells two more times with saponin buffer, resus suspend the pellet and 500 microliters of wash buffer for flow cytometric analysis.
In this figure H NPCs encapsulated and pure matrix are shown the cells grow in sphere, densely packed structures where the diameter of the spheres can be up to several hundred microns in between the spheres. Bundles of processes built up by the cells can be observed as indicated by the white arrows. Although individual cells can hardly be recognized, cells encapsulated in pure matrix supplemented with laminin grow in less dense structures and are more homogenously distributed as seen in this figure, single cells can even be recognized in less dense areas as indicated by the black arrows Here, H NPCs differentiated for four days in laminate supplemented pur Matrixx staining green for the expression of the neuronal marker.
Beta three tubulin are shown to evaluate the percentage of positive cells. The total cell number can be determined by enumerating cell nuclei stained here in blue with dappy. This micro photograph represents the full projection of a Z stack of pictures taken with a fluorescent microscope.
Cells released from the matrix can be seated again, for example, on cover slips as shown in this figure for further functional studies. This micro photograph shows cells cultured in 3D after being subjected to the previously mentioned release procedure and cultured for three days. The staining for beta three tubulin in red reveals a comparable morphology to the cells hosted in the 3D scaffold using unstained cells as a negative control.
The GA represented here as the black frame was set for the subsequent analysis of beta three tubulin positive cells in this dot plot positive cells appear within the black frame in the right part of the x axis and intermediate population in the dotted frame and most likely representing cell debris also can be observed. A comparison of manual cell counting and enumeration by flow cytometry is shown in this final figure. A much higher proportion of positive cells can be detected by flow cytometric analysis as indicated by the open black squares then can be typically enumerated by manual counting as indicated by the solid black triangles.
After watching this video, you should have a good understanding how to use poor metrics for cultivation of edge MPCs in 3D scaffold, and how to release the cells from the scaffold for flow cyto metric analyzers.
