Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

Summary

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone defects.

Abstract

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

Introduction

Mesenchymal stem cell (MSC) condensation is an essential stage to ensure the normal growth and development of the body in early organogenesis^1,2, especially in the formation of bone, cartilage, teeth, and skin^1,3,4. In the last few decades, tissue engineering therapies using cultured postnatal MSCs combined with biodegradable scaffolds have made important advances in osteogenic⁵ and cartilaginous regeneration⁶. However, the use of scaffolds may have some disadvantages, s....

Protocol

NOTE: All animal procedures were approved by the Animal Care and Use Committee of the Fourth Military Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Cryopreserved human UCMSCs that were obtained from a commercial source were used for the present study (see Table of Materials). The use of human cells was approved by the Ethics Committee of the Fourth Military Medical University. UCMSCs were taken as an example to describe the procedure. The cranial defect was taken as an example for showing the need of repair to describe the implantation procedure. All the experime....

Results

Aggregates can be successfully constructed from UCMSCs according to the experimental workflow (Figure 1). The quality of aggregates must be evaluated prior to use, via morphological observation and histological analysis. The lamellar structure formed should be complete and dense, with the cells interlaced to form a woven pattern by microscopic observation (Figure 2A). Edge curling can be discovered during aggregation; overcurling edges indicate unsucces.......

Discussion

With the advances of tissue engineering biotechnology, strategies to construct an implantable structure with high plasticity and containing long-term-surviving cells that can achieve optimal regeneration have been the focus of many scientists. There are a variety of current implantation methods of MSCs, such as cell-only methods, scaffolds complemented with cytokines^6,24, or the combination of stem cells and scaffolds⁵. This paper presents.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA (1x)	Sigma	T4049	Cell passage
Automatic Dehydration Machine	LEICA	ASP200s	Dehydrate aggregate
Centrifuge	Eppendorf	5418R	Centrifugation
Centrifuge tube	Thermo Nunc	339650	Centrifugation
Culture dish	Thermo	150466	Culture of UCMSCs
Ethanol	SCR	10009218	Dehydrate aggregate
Fatal bovine serum	Sijiqing	11011-8611	Culture of UCMSCs
Forcep	JZ	JD1080	Harvest aggregate
Glutaraldehyde	Proandy	10217-1	Fixation of aggregate
Hematoxylin and Eosin Staining Kit	beyotime	C0105S	HE staining
Hexamethyldisilazane	SCR	80068416	Dry aggregate surface
Hoechst33342	Sigma	14533	Cell nuclei stain
L-glutamine	Sigma	G5792	Culture of UCMSCs
Live/dead Viability/Cytotoxicity Kit	Invitrogen	L3224	Live/dead cell stain
Masson's Staining Kit	ZHC	CD069	Masson Staining
Minimum Essential Medium Alpha basic (1x)	Gibco	C12571500BT	Culture of UCMSCs
Paraffin	Leica	39601006	Tissue embedding
Paraformaldehyde	Saint-Bio	D16013	Fixation of aggregate
PBS (1x)	Meilunbio	MA0015	Resuspend and purify UCMSCs
Penicillin/Streptomycin	Procell Life Science	PB180120	Culture of UCMSCs
Pentobarbital sodium	Sigma	P3761	Animal anesthesia
Polysporin	Pfizer		Prevent eye dry
Scanning Electron Microscope	Hitachi	s-4800	SEM observation
Scissor	JZ	Y00030	Animal surgical incision
Six-well plate	Thermo	140675	Culture of UCMSCs
Stitch	Jinhuan	F603	Close wounds
Suture	Xy	4-0	Close wounds
Thermostatic equipment	Grant	v-0001-0005	Water bath
UCMSCs	Bai'ao	UKK220201	Commercially UCMSCs
Vitamin C	Diyibio	DY40138-25g	Aggregate inducing
Xylene	SCR	10023418	Dehydrate aggregate