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 resident cells. It is well-established that these signals can influence many fundamental cell behaviors, impacting their contractile function, proliferation, migration, and differentiation potential^7,8,9. Thus, ECM is increasingly being employed in tissue engineering and regenerative medicine as a therapeutic support tool^{9,10,11,12,13}.

The ECM consists of several proteins such as collagen, elastin, fibronectin, proteoglycan, and laminin, along with ECM-bound growth factors, all involved in regeneration mechanisms, such as cell recruitment, migration, and differentiation, as well as cell alignment and proliferation¹⁴. The mechanical properties of the tissues also have great relevance in the physiopathology of the organs. Indeed, changes in mechanical properties are often associated with the onset and the evolution of several diseases. The reason resides in the fact that when the ECM is modified, signals coming from the environment induce changes in gene and protein expression, leading to functional impairment^15,16.

Regenerative therapies for organ repair are currently focused on replicating the sophisticated microenvironment of the native tissue to heal the organ where the body fails. Despite the rapid pace of many tissue engineering approaches, the tissues still cannot be reproduced accurately in their entirety and complexity by artificial procedures. Synthetic materials have been largely employed so far, as they can be appropriately tuned to simulate the mechanical and biochemical properties of the cellular microenvironment. Nevertheless, they have limits, such as the inability to mimic the numerous interactions within the native tissue, the cost of technologies to produce them, and the fact that they are less natural and biocompatible than native tissue^17,18,19. Additionally, their composition, primarily in terms of proteins and soluble factors, greatly differs from the natural one, which is extremely difficult to replicate²⁰.

The cutting-edge approach in regenerative medicine to reduce the gap between the patients in need and organ transplants is to produce scaffolds made of decellularized extracellular matrix (d-ECM) and repopulate them with the appropriate cell types to regenerate the damaged organs. Decellularization is the process in which the ECM is isolated from its native cells and genetic material to produce a natural and biomimetic scaffold, able to avoid the immune response and rejection once implanted in the patients^21,22,23. The ECM thus obtained can then be repopulated to produce functional tissue. The major issue when developing a d-ECM is the method. For any decellularization technique, the primary goal remains the preservation of the native ECM composition, stiffness, and 3D structure, and all strategies have both benefits and drawbacks. Because the elimination of cellular content and DNA from the tissue requires the use of chemical or physical agents, or the combination of both, each decellularization procedure causes, to different degrees, the disruption of the ECM. Hence, it is crucial to minimize the damage to the ECM^24,25,26.

Native ECM utilization as a platform for the reconstitution of native ECM in vitro is highly desirable. For this purpose, several decellularization protocols have been applied to a wide range of tissues^27,28,29,30. In fact, since the early stages of decellularization research and ECM development, several tissues, such as arteries, aortic valves, and peripheral nerves from both animals and humans, have been decellularized, and some d-ECMs are still commercially available and used for tissue replacement or wound healing^31,32,33. Recently, human skin has also emerged as a suitable candidate to produce decellularized scaffolds for cardiac repair owing to its composition and mechanical properties-able to boost the regenerative potential of cardiac progenitor cells (CPCs) and adapt to cardiac contractility³⁴. This paper describes a simple and fast protocol to produce decellularized scaffolds from adult human skin, allowing the development of a d-ECM with well-preserved architecture.

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. Using a serological pipette, add 12 mL of Triton X-100 and a stir bar to the beaker. Place the beaker on a magnetic stirrer and mix the solution until the Triton X-100 is completely dissolved.
   2. Stop the agitation. Remove the stir bar using a stir bar retriever. Store at room temperature.
   3. Prepare 600 mL of 2% SDS solution by measuring 550 mL of double-distilled water in a graduated cylinder and transferring it to a 1 L beaker.
   4. Weigh 12 g of SDS powder in a plastic weighing boat and transfer it to the beaker containing the double-distilled water in step 1.1.3.
      NOTE: Step 1.1.4 must be performed under a chemical hood, and the user should wear personal protective equipment.
   5. Add a stir bar, place the beaker on a magnetic stirrer, and mix the solution until the SDS is completely dissolved.
   6. Stop the agitation process. Remove the stir bar using a stir bar retriever.
   7. Transfer the solution to a graduated cylinder and adjust the volume to 600 mL by adding double-distilled water.
   8. Pour 2% Triton X-100 solution prepared in step 1.1.1 into a 2 L cylinder.
   9. Pour 2% SDS solution prepared in steps 1.1.3-1.1.7 into the same 2 L cylinder to obtain a total volume of 1200 mL.
   10. Cover with parafilm and mix gently by inversion to obtain a homogeneous solution.
   11. Transfer the mixed solution to a 2 L bottle using a funnel to avoid the formation of foam. Store the solution at +4 °C.
2. Preparation of 1x phosphate-buffered-saline (PBS)
   1. Prepare 500 mL of 1x phosphate-buffered-saline (PBS) by dissolving 0.1 g of potassium phosphate monobasic, 0.1 g of potassium chloride, 4.0 g of sodium chloride, and 0.575 g of sodium phosphate dibasic in sterile double-distilled water. Check the pH value (7.4).
   2. Store at +4 °C until use.
3. Preparation of antibiotic solution
   1. Weigh 625 µg of amphotericin B in a plastic weighing boat.
   2. Pipette 8 mL of a penicillin and streptomycin (pen/strep) mixture into a 15 mL tube.
   3. Add the weighed amphotericin B to the pen/strep mixture, and adjust the volume to 10 mL by adding penicillin and streptomycin (pen/strep) mixture. Cap the tube and dissolve the amphotericin B by shaking vigorously.

2. Day 1 - start the decellularization procedure.

1. Pour 400 mL of the decellularizing solution into a 500 mL beaker.
2. Add 2.0 mL of the antibiotic solution.

3. Preparation of skin samples

1. Wash the human skin sample from the abdomen of patients undergoing abdominoplasty in a plastic tray by shacking and turning it upside down in a 0.9% NaCl isotonic physiological solution to remove excess blood and other biological fluids.
2. Remove hair using fine forceps and fat using large surgical scissors.
3. Place the skin sample on the dissecting board and dissect it using a scalpel, with the graduations on the dissecting board as a reference to obtain 3 cm x 2.5 cm fragments (length by width), avoiding scars and dirty or burned areas of the tissue.
4. Place each fragment in an embedding cassette, close it, and make sure all the cassettes are appropriately locked to avoid leakage of the sample during the decellularization procedure.
5. Place a maximum of four embedding cassettes with samples in a beaker with the decellularizing solution. Add a stir bar. Cover with aluminum foil, writing on the top all the information to identify the sample and the starting time of the procedure.
6. Place the beaker on the magnetic stirrer and start the agitation at a rotational velocity of 150 rpm. Stop the agitation. Remove the aluminum foil covering the beaker and take the embedding cassettes out with long forceps.
   NOTE: After stopping the agitation, check the clarity of the solution. Modify the duration of the agitation (less or more than 8 hours) based on the degree of the clouding due to the release of cellular debris.
7. Place each cassette in a 100 mm dish. Replace the decellularizing solution by repeating steps 3.5-3.6 overnight.

4. Day 2 - check the state of the skin samples.

1. Stop the agitation process. Remove the aluminum foil covering the beaker and take the embedding cassettes out with long forceps.
2. Place each cassette in a 100 mm dish and open it. Check the color of the sample.
   NOTE: The samples might show a color change from the native beige to snow-white.
3. Remove residual hair and detach the epidermis from the dermis. Replace the decellularizing solution by repeating steps 3.5-3.6.
4. Stop the agitation after 8 h. Repeat steps 4.1-4.2 until the sample appears snow-white. Stop the decellularizing procedure.
   NOTE: The time of exposure to the action of detergents can be extended if needed.
5. Measure 400 mL of 1x PBS in a 500 mL cylinder, pour it into a 500 mL beaker, and add 2 mL of the Pen Strep/Amphotericin B mixture. Add a stir bar.
6. Place a maximum of four cassettes containing the samples in each beaker with the 1x PBS/antibiotics solution. Cover with aluminum foil and write all the information on the top as in step 3.5.
7. Place the beaker on a magnetic stirrer and start the agitation at a rotational velocity of 150 rpm overnight, at room temperature.

5. Day 3 - a final wash of the samples

1. Stop the agitation. Replace the 1x PBS solution with 400 mL of double-distilled water. Add a stir bar. Cover with aluminum foil and write all the information on top as in step 3.5.
2. Place the beaker on a magnetic stirrer and start the agitation at a rotational velocity of 150 rpm for 30 min.
3. Stop the agitation process. Remove the aluminum foil covering the beaker and take the embedding cassettes out with long forceps.
4. Place each embedding cassette containing the decellularized skin sample in a 100 mm dish. Open the cassettes and gently dry the skin samples by dabbing them on laboratory wipes.
5. Place each decellularized skin sample in a weighing boat previously tagged with the information about the sample. Cover the boat with aluminum foil and write all the information on the top to identify the sample. Store at - 80 °C.

Results

The aim of the protocol was to obtain a skin d-ECM sample from biological tissue, maintaining a well-organized 3D structure and well-preserved content of biological molecules (Figure 1). This method is primarily based on the constant stirring of the samples in a solution containing the combination of two detergents, Triton X-100 and SDS, thus preserving the biological and structural features typical of the native tissue and reducing the time of exposure during the decellularization process. ...

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