Het optimaliseren van Bevestiging van menselijke mesenchymale stamcellen op Poly (ε-caprolacton) elektrogesponnen Yarns

In this procedure, electro spun PCL scaffolds are set up in different ways to determine an optimal cell seeding setup. First, the sterile scaffolds are placed in the different setups being investigated, including cell culture inserts, troughs, and bioreactor rotary vessels. Once in place, a known number of cells are seeded onto the scaffold and then left undisturbed for 20 minutes.

Then all samples are carefully moved to an incubator, set up 5%carbon dioxide and 37 degrees Celsius, and subjected to static or dynamic conditions before hours. Following this culture time, the number of cells that have attached to the scaffold is determined by A DNA assay to quantify the amount of cells in the scaffold media and well fractions. Ultimately, this is achieved by quantifying the DNA present on the scaffold, the well and in the surrounding media to view the attachment of cells to scaffolds scanning electron microscopy or SEM is used.

We first had the idea for this investigation when we knew our scaffolds didn't cover the entire base of the well plate, which meant there was a possibility that not all of the cells would be in contact with the scaffold and hence able to attach. To begin, set up the cell culture inserts under lamina flow by opening the sterile six well cell culture inserts and separating the shorter rings with teeth from the wider ringed bodies. Then take the ring with the teeth pointing upwards and drape one of the four centimeter scaffolds over the center of the ring, making sure it overlaps on both sides.

Take the ringed body and position it over the tooth ring and scaffold. Then push downwards, making sure the scaffold stays in position and lies through the center of the cell culture.Insert. Place the cell culture insert with scaffold into the well of a six well low binding plate, and add 10 milliliters of culture media to the scaffold.

Next place polytetrafluoroethylene troughs into individual Petri dishes and add 10 milliliters of culture media to the trough. Using forceps drape one of the three centimeter scaffolds into the trough, making sure it's length lies parallel to the trough's longer edge. To set up the bioreactor vessel under lamina flow, dispense 10 milliliters of sterile phosphate buffered saline or PBS through the bioreactor vessel's main port.

After 10 minutes, remove the PBS and replace with eight milliliters of culture media using forceps. Insert one of the three centimeter scaffolds into the vessel via the main port. Then pose off the main port.

After performing human mesenchymal stem cell counting as described in the text protocol, aspirate the media from the centrifuge tube leaving the cell pellet and replace with the calculated media volume. Resus suspend the cells and media for an even mix. Using a P 200 Gilson pipette slowly dispense 200 microliters of cell suspension into each scaffold by running the tip of the pipette along the scaffold length and below the media liquid surface.

Leave undisturbed for 20 minutes, or the bioreactor vessels dispensed 200 microliters of the cell suspension through the main port. Top up the remaining two milliliters of culture media by the syringe ports to give a total volume of 10 milliliters. Next, transfer the bioreactor vessels to the rotary vessel bioreactor, and set to rotate.

At nine RPM, transfer the well plates with cell culture inserts and troughs to the shaker plate and set to rotate at 30 RPM in a 37 degrees Celsius, 5%carbon dioxide incubator or the static culture. Transfer the well plates with the cell culture inserts and troughs to the shelf of a cell culture incubator. Set at 37 degrees Celsius and 5%carbon dioxide.

After four hours, remove the samples from the incubator and place under lamina flow. Next, remove the media fractions from all samples and place in separately labeled centrifuge tubes. Following centrifugation for five minutes at 241 times G, remove the supinate before adding three milliliters of lysis buffer vortex, the solution to break up the cell pellet the scaffolds held within cell culture inserts First, free the scaffold by cutting the scaffold close to the inserts edge using a scaffold.

Then using forceps, remove the scaffolds and place in centrifuge tubes containing three milliliters of lysis buffer. Then add three milliliters of lysis buffer to each scaffold receptacle and scrape the surface. Remove the lysis buffer and place in separately labeled centrifuge tubes.

Vortex each centrifuge tube for approximately one minute to ensure sufficient agitation and to encourage lysis of the cell membrane. In a black 96 well plate add 100 microliters of lysis buffer for each sample fraction scaffold media, and well then in the dark, add 100 microliters of cell DNA solution to all wells containing lysis buffer and mix. Gently include wells with lysis buffer containing no DNA and cell DNA solution to provide a negative and positive control for the well plate.

Using a fluorescence plate reader, measure the absorbance of the wells at 485 nanometer excitation and 520 nanometer emission. Compare the data with the standard curve generated from the DNA standards as per the manufacturer's instructions to perform SEM fixation. After four hours of incubation, remove all scaffolds from their receptacles and place within separate wells of a new six well plate and a lamina flow.

Then wash the scaffolds twice with PBS to each. Well add two milliliters of 1.5%glutaraldehyde in PBS to ensure complete coverage at the scaffold. Leave the plate for a minimum of 30 minutes at four degrees Celsius for cel fixation.

Remove the fixative solution and wash the scaffolds twice with PBS. Then transfer the scaffolds to a new well plate to dehydrate them with increasing concentrations of ethanol in distilled water, starting with 50%ethanol followed by 70%and then 90%for each concentration. Fully submerse the scaffolds in solution and leave for three minutes before discarding the solution and repeating.

Once dehydrate the scaffolds in 100%ethanol by fully submerging the scaffolds in solution and leaving for five minutes, discard the solution and repeat chemically dry the scaffolds. Using HMDS within a fume cupboard. Immerse the scaffolds in HMDS and leave for five minutes before removing the HMDS after repeating once.

Remove the HMDS and allow the scaffolds to dry. Then mount the scaffolds on commercially available SEM stubs to ease viewing within the SEM coat, the samples with a gold spotter coat for two minutes to ensure a thin and even coverage. Finally, place the samples within the SEM and visualize the cell seated scaffolds using a five kilo electron vault electron beam.

The results highlight the location of cells following four hours post seeding for each experimental setup investigated. This figure demonstrates the percentage of cells that have attached to the scaffold surface during this time where all seeding setups investigated. The percentage of cell attachment is relatively low, but the greatest cell adherence for scaffolds held within cell culture inserts and shaken at 30 RPM.

The lowest adherence was for scaffolds held within the cell culture inserts and kept under static conditions. A large number of cells were present within the media fraction, most notably for cell culture. Inserts held within low binding plates at 50%and rotary vessels at 51%Scaffolds held within the troughs demonstrated a raised number of cells present within the holder itself.

48%for trough shaker and 50%for trough static scanning. Electro microscopy allowed a visual assessment of the cell seeded scaffolds. Representative images highlighted a limited presence of cells on the fibrous surface irrespective of seeding setup.

However, a greater number of cells and cell agglomerates were present on the scaffolds held within cell culture inserts and shaken at 30 RP M.After watching this video, you should have a good understanding that seeding a known number of cells did not necessarily equate to that many cells actually attaching to the scaffold, especially for scaffolds that do not cover the entire base of the wild plates. For scaffolds such as these, it is recommended that different cell seeding setups are first optimized in order to maximize initial cell attachment to the scaffold.