Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy has been widely reported as a promising treatment for many diseases. And the cell sheet techniques have been developed in order to improve the cell's retention and the survival within the targets, too. During cell sheets construction, the insufficient nutrition supply remain a key problems for the loss of stem cell's function, and is an unsatisfactory stem cell property would finally effect the therapeutic effects in vivo.

Our purpose to prepare available multilayered stem cell sheet product, our team developed the cellularized polesized, pericardium and cell sheet scaffold. Based on the scaffold the multilayer cells starter would be quickly constructed with nonapeptide Hydrogen and a dynamics perfusion system is used to ensure the stem cell's nutrition supply in vitro. Take out a dried DBP scaffold and put it in the center of the black base.

Put a white tension ring on the DBP scaffold, and affix it in the tissue carrier. Ensure the DBP scaffold is totally fixed in the tissue carrier. Add 100 microliters culture mediums on the DBP scaffold.

Put the scaffold into the 37 degree Celsius incubator, and allow it to soak for 15 minutes. Take the cells out of the incubator, remove the culture mediums from the culture dish, gently wash the cells with two milliliters of warm PBS. Remove all PBS from the culture dish, and make sure no liquid remains.

Add two milliliters to 1.25%trypsin solution to the dish, and incubate the cells for three minutes. Stop the trypsin effect by adding two milliliters culture medium. Gently wash the cells from the dish.

Transfer the cell suspensions in a new 15 milliliter centrifugal tube. Centrifugate the cells at 220 times gravity for five minutes, and obtain cell sediments. Remove the supernatant, and wipe out the cell sediment.

Re suspend the cells with three milliliter 10%sucrose solution. Aspirate 10 microliters cell suspension, and the content cell number reads a hemocytometer, extract three million cells and transfer in your new 1.5 milliliter centrifugal tube. Centrifugate the cells at 220 times square root for five minutes, completely remove the supernatants and obtain the cells sediment.

Prepare 20 microliters 10%sucrose solution. Gently re-suspend the cells and obtain a uniform suspension. Add 20 microliter peptide hydro gel at the top of suspension gently mix the peptide hydro gel, and the cells suspension with the T, take out the DBP scaffold, leaving the tissue carrier, and then gently aspirate the calculi median with a T.Aspirate the mixture the mixture and evenly add on to the DBP scaffold.

Add one milliliter calciumedetate to the bottom of the tissue carrier. Gently add four milliliter cultural medium in the culture dish and then emerge the cell sheet. Put the cells in the incubator for two hours let it culture.

The dynamic perfusion system include a perfusion culture container the gas exchanging recrement and a glass bottle. Assembles are dynamic perfusion systems as the figure shown. Add two hydro milliliters culture medians in the glass bottle.

Insert the cell sheet into the chamber of the culture container, add three milliliters culture medians in the container, put the dynamic perfusion systems in the incubators, and start to pump. Open the culture container in the bipositive recumbent. Take out the cell sheet from the culture container and put it in culture dish.

Use one forceps to immobilize the tissue carrier and use another two forceps to separate the white tension ring from the black base. Finally, obtain the multilayered cell sheet. DBP scaffold is transparent.

The SEM resells shows that colleges captured and the components are completely reserved. The cell sheets can be obtained and held by forceps for short preservation. These cell sheet could be transferred to 1.5 milliliter cylindriform tube.

As a model for instance starting with cell showings of BMFC'S highly positive for the stem cells marker CD 9O and the CD 29. After cell construction the BMFC'S leaving the cell sheet remains high expansion levels of CD 90 and the CD 29. Constructing the multilayered cell structure is the critical step of the protocol.

The pan time requires four hydro gel when the PH value changes from SA to neutral, therefore, it is important to wash the cell with sucrose solution to remove the surface ions. Also, the volume ratio of cell suspension and panthihydrogel is not good friend for constructing the temporary multilayer structure. After the formation of 3D stem cell structure, you will see the dynamic perfusion system is important for stabilizing the multilayered structure as well as maintaining the stem cell viabilities.

The dynamic infiltration of the culture median could facilitate stem cells to proliferate and extrapolate cell context within the multilayered structure also the dynamic culture thermometers should be adjusted according to different stem cell types.