This work was supported by the National Natural Science Foundation of China (grant number 31771064); the Science and Technology Planning Project of Guangdong Province (grant numbers 2013B010404030, 2014A010105029, and 2016A020214012); the Science and Technology Planning Project of Guangzhou (grant number 201607010063); and the Undergraduate Innovation and Entrepreneurship Training Program (grant number 201610559028); the National Science Foundation for Young Scientists of China (grant number 31800819).

All stem cell and animal experiment procedures were conducted according to the ethical guidelines of the National Guide for the Care and Use of Laboratory Animals and approved by the Jinan University Animal Care and Use Committee (Guangzhou, China).
1. Preparation of the DPP Scaffold with the PLA2 Decellularization Method14
Note: See Figure 1A for a schematic of the PLA2 decellularization meth...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+scaffold+provides+a+cell+delivery+platform+for+cardiovascular+repair.">Composite scaffold provides a cell delivery platform for cardiovascular repair.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (19), 7974-7979 (2011).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+sheets+of+collagen/elastin/polycaprolactone+improve+cardiac+repair+after+myocardial+infarction.">Electrospun nanofibrous sheets of collagen/elastin/polycaprolactone improve cardiac repair after myocardial infarction.</a> American Journal of Translational Research. 8 (4), 1678-1694 (2016).</li><li>Arana, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+delivery+of+collagen+patches+with+adipose-derived+stem+cells+in+rat+and+minipig+models+of+chronic+myocardial+infarction.">Epicardial delivery of collagen patches with adipose-derived stem cells in rat and minipig models of chronic myocardial infarction.</a> Biomaterials. 35 (1), 143-151 (2014).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+high+bioactivity+multilayered+bone-marrow+mesenchymal+stem+cell+sheets+for+myocardial+infarction+using+a+3D-dynamic+system.">Preparation of high bioactivity multilayered bone-marrow mesenchymal stem cell sheets for myocardial infarction using a 3D-dynamic system.</a> Acta Biomaterialia. 72, 182-195 (2018).</li><li>Wu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+phospholipase+A(2)+to+prepare+acellular+porcine+corneal+stroma+as+a+tissue+engineering+scaffold.">The use of phospholipase A(2) to prepare acellular porcine corneal stroma as a tissue engineering scaffold.</a> Biomaterials. 30 (21), 3513-3522 (2009).</li><li>Degano, I. 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The present protocol reports an efficient method for constructing a multilayered MSC sheet. This cell sheet exhibits optimal mechanical strength, high cell seeding density, and favorable stem cell bioactivity. Using BMSCs as an example, the 3D cell structure is quickly constructed with RAD16-I peptide hydrogel. After being cultured in the dynamic perfusion system, the multilayered BMSC sheet is successfully obtained and the BMSCs maintain a high expression of stem cell markers.
Constructing th...

Stem cell therapy has been reported as an effective treatment for many diseases; however, low cell retention and poor survival within the target zone remain critical issues following traditional stem cell injection. To solve this problem, tissue engineering scientists developed the cell sheet technique. A monolayered cell sheet with intact extracellular matrix was firstly prepared by using the temperature-response culture dish1, and its follow-up studies reported the significant improvements of stem cell retention and survival within the infarcted area2,3. Among the methods, constructing the multilayered cell sheet has been reported as an effective strategy for improving the cell survival and the cell sheet therapeutic effect3,4. Since then, scientists have worked on developing different cell sheet construction methods in order to increase the cell amount, stem cell property, and mechanical property of the cell sheets. So far, certain types of cell sheet have been constructed and studied in the treatment of myocardial infarction5, cartilage injury6, and skin wound7.
The bioactivity of stem cells before transplantation showed an emerging influence on injured tissue regeneration, and different cell sheet construction strategies have different effects on the stem cells. On one hand, confluent cell sheets only consisted of high-density stem cells, and natural extracellular matrices could be acquired by stacking monolayered cell sheets8 or by using magnetic tissue engineering techniques9. On the other hand, researchers developed different scaffolds to provide adequate mechanical strength and support cell growth10,11,12, which allowed a low stem cell seeding density to ensure the nutrition supply. However, despite these approaches, the low efficient nutrition supply within the multilayered cell sheet structure remains a major concern during the in vitro construction. Therefore, an efficient and feasible cell sheet construction system is urgently required.
This protocol describes the steps to prepare a multilayeredmesenchymal stem cell (MSC) cell sheet. In this construction system, the cell sheet mechanical strength is provided by a DPP. Based on this scaffold, the 3D cell structure can be quickly constructed with RAD16-I peptide hydrogel, and a dynamic perfusion system is used to culture the multilayered cell sheet, in order to stabilize the 3D cell sheet structure and provide sufficient nutrition supply for the cells. Using this system, a multilayered BMSC sheet was successfully prepared and exhibited an optimal therapeutic effect on the rat myocardial infarction model13.

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			<td style="width:167px;">354250</td>
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			<td height="38" style="height:39px;width:216px;">Dynamic perfusion culture system</td>
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			<td style="width:167px;">IPC N8</td>
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			<td height="21" style="height:21px;width:216px;">Pump tubing</td>
			<td style="width:108px;">Ismatec</td>
			<td style="width:167px;">Nr.1306</td>
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			<td style="width:108px;">Regensburg</td>
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			<td>tissue carrier components&#160;</td>
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			<td height="21" style="height:21px;">MINUSHEET</td>
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			<td>gas exchange equipment&#160;</td>
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			<td height="21" style="height:21px;">MINUSHEET 0002</td>
			<td style="width:108px;">Regensburg</td>
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			<td>500 mL glass bottle&#160;</td>
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			<td height="21" style="height:21px;">MINUSHEET 1301</td>
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			<td style="width:167px;"></td>
			<td>perfusion culture container&#160;</td>
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construction of a multilayered mesenchymal stem cell sheet with a 3d dynamic culture system

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

The schematic of the multilayered stem cell sheet construction is shown in Figure 1. Preparing the cell sheet scaffold by the PLA2 decellularization method is the first step. Based on the scaffold, a temporary 3D cell structure is constructed by mixing the stem cells with the RAD16-1 peptide hydrogel. In order to obtain a multilayered cell sheet with favorable stem cell bioactivity and optimal mechanical strength, the cell sheet is cultured in a dy...

Watch this Scientific Journal Video about Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System at JoVE.com

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

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

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.

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7.4K visualizações.  Jinan University. A terapia com células-tronco tem sido amplamente relatada como um tratamento promissor para muitas doenças. E as técnicas de folha de celular foram desenvolvidas para melhorar a retenção da célula e a sobrevivência dentro dos alvos, também. Durante a construção de folhas de células, o suprimento nutricional insuficiente continua sendo um problema fundamental para a perda da função das células-tronco, e é uma propriedade insatisfatória de células-tronco que finalmente afetaria...