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

Summary

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

Abstract

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.

Introduction

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 area^2,3. Among the methods, constructin....

Protocol

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 method.

1. Prepare 100 mL of 200 U/mL PLA₂ solution. Add 0.5 g of sodium deoxycholate and 2 mL of PLA₂ in 198 mL of carbo....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phospholipase A2	Sigma-Aldrich	P6534
Sodium deoxycholate	Sigma-Aldrich	D6750-100G
Phosphate buffer	Gibco BRL	89033
Penicillin streptomycin / amphotericin	Gibco BRL	15640055
Buffer bicarbonate	Sigma-Aldrich	C3041
Table concentrator	Changzhou Aohua Instrument Co.	KT20183
Dulbecco's Modified Eagle Medium(DMEM)	Corning Cellgro	10-014-CVR
South American fetal bovine serum	Gibco BRL	10270-106/P30-3302
L-Glutamine	Corning Cellgro	25-005-CI
0.25% Trypsin/2.21 mM EDTA	Corning Cellgro	25-053-CI
Biosafety cabinet	Esco,Singapore	AC2-2S1
Constant temperature incubator	Esco,Singapore	CLS-170B-8
Centrifuge tube	Corning	430790
EP tube	Axygen	31617934
Centrifugal machine	TOMOS	1-16R
Sucrose	Sigma-Aldrich	S9378-500G
Pura Matrix	BD	354250
Dynamic perfusion culture system	Minucells and Minutissue	D-93077
Peristaltic pump	Ismatec	IPC N8
Pump tubing	Ismatec	Nr.1306
MINUSHEET 1300	Regensburg		tissue carrier components
MINUSHEET	Regensburg		dynamic perfusion system
MINUSHEET 0006	Regensburg		gas exchange equipment
MINUSHEET 0002	Regensburg		500 mL glass bottle
MINUSHEET 1301			perfusion culture container