Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Summary

Here, we present a protocol to develop an in vivo cancer model using cell sheet technology. Such a model could be very useful for the evaluation of anticancer therapeutics.

Abstract

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.

Introduction

HCC is a primary cancer of the liver that is significantly associated with a poor prognosis. Annually, almost a half million new patients are diagnosed with HCC, representing 85% of liver cancer patients worldwide¹. Hepatocarcinogenesis is not a single-form disease; rather,it is a collection of diseases that have different histopathological features and genetic and genomic variability, in addition to varied prognostic outcomes¹. Therefore, the main challenges in the development of an effective therapeutic strategy for HCC are the limited knowledge of HCC biology and the lack of a suitable experimental animal mo....

Protocol

The protocol follows the animal care guidelines of King Saud University's ethical committee. Surgical procedures, anesthetics, and other medications used on animals are approved by King Saud University's ethical committee. All experimental work is performed by appropriately trained personnel.

1. Cell Sheet Construction

1. Coat the culture dishes (3.5-cm temperature-responsive culture dishes) with undiluted fetal bovine serum (FBS) and ensure to cover all the surface of the dish.
   NOTE: This step is important for the detachment of the cell sheet since it supports the growth of the cells in a monolayer and prevents cell....

Results

Tumorigenicity of Transplanted Cell Sheets in Rats:

One month after the transplantation, all transplanted cell sheets in rats' livers have developed tumors (Figure 3). The average size of the developed tumors from HepG2, HepG2/BMMSC, and HepG2/UCMSC cell sheets were 4.5 cm, 4 cm, and 2.5 cm, respectively¹⁰.

Discussion

An extensive amount of research is dedicated to developing an adequate in vivo preclinical animal model that resembles human cancers. Currently, the major approaches used to create cancer animal models involve genetic engineering and cell transplantation¹¹. Genetically modified animal models are good tools for the identification and validation of target genes, as well as understanding the molecular mechanisms underlying drug-induced toxicity. However, this model is associated with some li.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
FBS	Gibco/Invitrogen	10270106
DMEM high glucose	Sigma	D5671-500ML
Penicillin/streptomycin	Life Technology	15070063
Sterile physiologic saline	Sigma	S0817-1GA
Human HepG2 cell line	ATCC, USA	HB-8065
Human bone marrow MSCs cell line	PromoCell, USA	C-12974
human umbilical cord tissue MSCs	PromoCell, USA	C-12971
Ketamine 50%	Rompun, Bayer
Xylazine 2%	Rompun	23076-35-9
Alphadine® solution.	Riyadh Pharma	LBL0816
Disposables:
15mL Polypropylene High Clarity PP Centrifuge Tube	Falcon	352097
3.5 cm sterile UpCell culture dishes with the filter paper (membrane)	Sigma	174904-1CS
100-1000 µl  Pipette Tips	Sigma	CLS4868-1000EA
Basic Procedure Drape	Thermofisher	PMD5293.0
Equipment
Plus pipette, variable volume	Eppendorf® Research®	Z683779-1EA
Tissue culture incubator 37 °C, 5% CO2	Any brand
Biological safety cabinet	Any brand
Tissue culture incubator 20 °C, 5% CO2	Any brand
Sterile surgical tools and nude rats:
Forceps
Scissors
scalpel
Nylon Suture  5-0	Accutome	AB-3854S	Monofilament, Lancet
1 ml Tuberculin Syringes	Fisher Scientific	14-826-88
Nude rats	Charles river