Name: construction of a multilayered mesenchymal stem cell sheet with a 3d dynamic culture system
Uploaded: 2018-10-20T15:00:00
Description: Watch this Scientific Journal Video about Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System at JoVE.com

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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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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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 ...

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