In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

Summary

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

Abstract

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced “in vivo-like” model for hypertrophic scar-related studies.

Introduction

Scar tissue represents the endpoint of tissue repair. However, in many individuals, especially those suffering from burns or trauma¹, scarring can be excessive and impose undesirable effects on the morphology and functioning of healed skin. Although the exact mechanisms of pathological (hypertrophic scars and keloids) scar formation are not fully understood, excessive deposition of collagen during wound healing has been demonstrated to be an essential contribution².

It is well-established that transforming growth factor beta 1 (TGF-β1) and alpha smooth muscle actin (αSMA) play key ro....

Protocol

1. Cell culture

1. Maintain hHSFs and normal dermal fibroblasts derived from non-pathological tissue (hNSFs) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and 1% v/v penicillin/streptomycin solution (P/S) at 37°C in an incubator with 5% CO₂/95% air.
2. Purchase Ficoll 70, Ficoll 400, and ascorbic acid from the appropriate companies.

2. Construction of MMC hypertrophic scar model

1. Seed the hHSFs or hNSFs (50,000/well) into a 24 well plate containing 1 mL of media in each well.
2. Place in a 37°C incubator at 5% CO₂ and leave ....

Results

Triplicate samples were performed in each experiment, and each experiment was repeated 3x using cells from three individual patients. Data are expressed as percentages of the control group. One-way ANOVA and Tukey's post-hoc test were applied to analyze statistical differences (*p ˂ 0.05).

MMC using Ficoll at 9% FVO (fractional volume occupancy) enhances the total amount of collagen and collagen I deposition in hHSF¹³. As illustrated in Fig.......

Discussion

This protocol aims to optimize and authenticate an improved :scar-in-a-jar” in vitro model for human cutaneous scar tissue. Previous studies have reported the application of MMC technique to human lung fibroblasts¹², human bone marrow mesenchymal stem cells²³, and human dermal fibroblasts²³ using dextran¹², Ficoll¹², and PVP²³ as crowders. In the study reported here, the pr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 μm filter	Sartorius	16534
2-Mercaptoethanol	Sigma-Aldrich	M6250
4’,6-diamidino-2-phenylindole (DAPI)	Thermo Fisher Scientific	P36962
Alexa Fluor 680	Thermo Fisher Scientific	A-21076
Alexa Fluor 800	Thermo Fisher Scientific	A-11371
alpha smooth muscle actin (αSMA) primary antibody	Abcam	ab5694
Applied Biosystems 7500 Fast Real-Time PCR System (thermal cycler )	Thermo Fisher Scientific	4351106
Ascorbic acid	Wako	#013-12061
Bovine serum albumin	Sigma-Aldrich	#A2153
Bradford protein assay	Bio-Rad	500-0006
Collagen I primary antibody (for immunostaining)	Abcam	6308
Collagen I primary antibody (for western blot)	Abcam	ab21286
Collagen III primary antibody	Abcam	ab7778
Collagen IV primary antibody	Abcam	ab6586
Direct Red 80	Sigma-Aldrich	2610108
Dulbecco's Modified Eagle's Medium (DMEM)	Life Technologies	11996-065
Fetal calf serum (FCS)	Life Technologies	6000-044
Ficoll 400	GE HealthCare	#17-0300-10
Ficoll 70	GE HealthCare	#17-0310-10
GAPDH primary antibody	Sigma-Aldrich	G8795
Goat Anti-Rabbit secondary antibody	Abcam	ab97050
Human hypertrophic scar/normal fibroblasts (hHSF/hNSF)	Cell Research Corporation	106, 107, 108
iScript cDNA Synthesis Kit	Bio-Rad	#1708890
MMP-1 primary antibody	Abcam	ab38929
MMP-13 primary antibody	Abcam	ab39012
MMP-2 primary antibody	Abcam	ab37150
MMP-9 primary antibody	Abcam	ab38898
NanoDrop Microvolume Spectrophotometers	Thermo Fisher Scientific	N/A
Nitrocellulose membrane	Bio-Rad	10484060
NuPAGE 4-12% Bis-Tris Protein Gels	Thermo Fisher Scientific	NP0321BOX
Odyssey blocking buffer	LI-COR Biosciences	927–40000
Odyssey Fc Imaging System	LI-COR Biosciences	N/A
Olympus IX-81 HCS microscope (for immunostaining)	Olympus	N/A
Penicillin/streptomycin solution (P/S)	Life Technologies	15140-122
PrimePCR Assays	Bio-Rad		Customized primers pre-coated in 96-well plates based on requirement
Protease inhibitor cocktail (PIC)	Sigma-Aldrich	11697498001
PVP 360	Sigma	#PVP360
PVP 40	Sigma	#PVP40
RIPA buffer	Merck	R0278
RNeasy Plus Mini Kit	QIAGEN	#74134
Sodium vanadate	Sigma-Aldrich	450022
Sodium vanadate	Sigma-Aldrich	450243
SpectraMax M5 Multi-Mode microplate reader	Molecular Devices	N/A
SsoAdvanced universal SYBR green supermix	Bio-Rad	#172-5270
Tween 20	Sigma-Aldrich	P9416