Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Summary

Decellularized human skin is suitable for tissue regeneration. A major issue of decellularization is the preservation of the native architecture, along with the appropriate content of structural proteins, glycosaminoglycans (GAGs), and growth factors. The method proposed allows fast and effective decellularization, producing decellularized skin with well-preserved native features.

Abstract

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells.

The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

Introduction

The ECM serves as a scaffold for cells, supporting them through an intricate architecture maintained by different components, and it is one of the major factors responsible for the mechanical properties of the heart and cardiac tissue function^1,2. Increasing evidence suggests that ECM plays an active role in tissue remodeling, making the conventional assumption that the ECM is a passive component obsolete^3,4,5,6. The role of the ECM is to provide biophysical and biochemical cues to ....

Protocol

The specimens from human tissue were collected according to the principles of the Declaration of Helsinki and observing University Hospital "Federico II" guidelines. All patients involved in this study provided written consent forms.

1. Preparation of solutions

1. Preparation of 1200 mL of 1% decellularizing solution
   1. Prepare 600 mL of 2% Triton X-100 solution by measuring 588 mL of double-distilled water in a graduated cylinder and transferring it to a 1 L beaker. .......

Discussion

Although the protocol described above has been optimized and improved compared to previously published protocols, it presents a few critical steps that need attention and precision. The formation of foam must be avoided during the preparation of the decellularizing solution to prevent incorrect dilution of the detergents. This could be addressed by gently pouring the solutions and making them flow along the inner side of the cylinder. Furthermore, care must be taken when manually removing fat tissue from the samples, as .......

Materials

Name	Company	Catalog Number	Comments
0.9% NaCl isotonic Physiological solution	Sigma-Aldrich	S8776	0.9% in water
1 L beaker	VWR	511-0318	Clean and autoclave before use
10 mL serological pipet	Falcon	357551	Sterile,  polystyrene
100 mm plates	Falcon	351029	Treated, sterile cell culture dish
15 mL sterile tubes	Falcon	352097	Centrifuge sterile tubes, polypropylene
1 L graduated cylinder	VWR	612-1524	Clean and autoclave before use
2 L bottle	VWR	215-1596	Clean and autoclave before use
25 mL serological pipet	Falcon	357525	Sterile,  polystyrene
2 L graduated cylinder	VWR	612-3072	Clean and autoclave before use
500 mL beaker	VWR	511-0317	Clean and autoclave before use
Amphotericin B	Sigma-Aldrich	Y0000005	Powder
Dissecting board	VWR	100498-398	Made of high-density polyethylene.
Dissecting scalpel	VWR	233-5526	Sterile and disposable
Embedding cassettes	Diapath	070191	External dimensions: 40x26x7 mm (WxDxH)
Fine forceps	VWR	232-1317	Clean and autoclave before use
Funnel	VWR	221-1861	Clean and autoclave before use
Hexagonal weighing boats size M	Sigma-Aldrich	Z708585	Hexagonal, polystyrene, 51 mm Bottom I.D., 64 mm Top I.D.
Hexagonal weighing boats size S	Sigma-Aldrich	Z708577	Hexagonal, polystyrene, 25 mm Bottom I.D., 38 mm Top I.D.
Large surgical scissors	VWR	233-1211	Clean and autoclave before use
Long forceps	VWR	232-0096	Clean and autoclave before use
Penicillin and Streptomycin	Sigma-Aldrich	P4333-100ml	Stabilized, with 10.000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered. Store at -20°C.  The solution  should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles Solution.
Pipette gun	Eppendorf	613-2795	Eppendorf Easypet® 3
Plastic tray	VWR	BELAH162620000	Corrosion-proof polypropylene plastic tray
Potassium Chloride	Sigma-Aldrich	P9333	Powder
Potassium Phosphate Monobasic	Sigma-Aldrich	P5665	Powder
Sodium Chloride	Sigma-Aldrich	S7653	Powder
Sodium Dodecyl Sulfate	Sigma-Aldrich	62862	Powder
Sodium Phosphate Dibasic	Sigma-Aldrich	94046	Powder
Spatula	VWR	RSGA038.210	Clean and autoclave before use
Spoon	VWR	231-1314	Clean and autoclave before use
Stir bar	VWR	442-0362	Clean and autoclave before use
Stir bar retriever	VWR	89026-262	Molded in pure, FDA-approved PTFE
Triton X-100	Sigma-Aldrich	9002-93-1	Liquid