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Decellularization of Bovine Small Intestinal Submucosa
Decellularization technology promises to overcome some of the significant limitations in the regenerative medicine field by providing functional biocompatible grafts. The technique involves removal of the cells from the biological tissues or organs for further use in tissue engineering and clinical interventions. There are significant differences between decellularization protocols due to the intrinsic properties of different tissue types and purpose of use. This multistep, chemical-solution-based protocol is optimized for the preparation of decellularized bovine small intestinal submucosa (SIS).
Keywords:Decellularization Bovine Small intestinal submucosa (SIS) Xenogenic biomaterials Extracellular matrix (ECM) Regenerative medicine Bioactive materials
With the advent of decellularization techniques, last 10 years have witnessed a new impetus in the field of biomaterials, namely for the production of extracellular matrix (ECM) based scaffolds. These studies initiated a new stage that will enable the use of biological (xeno-, allo-, and auto-) ECM for clinical tissue engineering/regenerative medicine applications. The basic principle of the decellularization technique is to remove the cells from a tissue or organ from various sources to obtain a biocompatible natural three-dimensional structure [1, 2]. A body of work shows that upon decellularization, ECM-based biomaterials retain collagen types, glycosaminoglycans, and growth factors such as transforming growth factor-β (TGF-β), basic fibroblast growth factor (b-FGF), and vascular endothelial growth factor (VEGF) that play active roles in regeneration by contributing to the cell behavior [3, 4, 5, 6, 7]. Various tissue types including small intestinal submucosa (SIS), heart valve, skin, tendon, bladder, blood vessel, skeletal muscle, and others have been used in preclinical tissue engineering studies; thus some of them have been translated into clinical use [7, 8]. Among all, the SIS layer has attracted considerable attention, thanks to its rich ECM content and ease of procurement. Decellularized SIS is an ECM-based biomaterial, which is commonly isolated from porcine small intestine. It has been approved by the FDA for treating venous ulcers, diabetic ulcers, skin injuries, also for repairing bladder wall, hernia, vascular defects, and lastly cartilage regeneration [7, 9, 10, 11, 12, 13]. Chemical agents employed for decellularization of biological materials show variation, depending on the purpose of use and tissue source. Therefore, the decellularization protocol must be assessed by considering the structure and cellular content of the intended tissue or organ sample. For example, the effectiveness of the chemicals would possibly vary between tendon and liver decellularization [14, 15, 16]. In addition to cellular composition, the thickness, the compactness, the lipid composition, etc. of a tissue or organ will define the decellularization technique and choice of solutions . Most decellularization protocols are multistep protocols involving physical, chemical, and additionally enzymatic digestion steps [17, 18]. Generally, physical methods such as agitation, sonication, electroporation, high pressure, or freezing/thawing comprise the initial step of the decellularization protocols. This stage aims to disrupt the cell membrane to release cellular components. Washing process removes the majority of the cellular components. However, physical methods are often not sufficient; hence, the efficiency of decellularization protocol is improved by the use of chemical or enzymatic agents [7, 19, 20, 21]. In this chapter, we provide an effective multistep protocol that utilizes physical and chemical agents for the decellularization of bovine SIS layers.
All solutions should be prepared with analytical grade reagents in ultrapure water (by purification of deionized water to obtain 18 MΩ-cm sensitivity at +25 °C). Unless otherwise indicated, prepare all reagents at room temperature and store at +4 °C until use. All the steps of isolation and decellularization should be carried out by shaking at room temperature. Waste disposal regulations should be carefully followed at all times.
Shaking water bath
Ultralow temperature freezer (−86 °C)
Liquid nitrogen tank
Surgical scissors, clamps, and forceps
Glass conical flasks and beakers in various volumes
2.2 Bovine Small Intestinal Submucosa Isolation and Decellularization Agents
Isotonic sodium chloride (NaCl) solution: Add 9 g NaCl to 1 L of distilled water inside an Erlenmeyer flask and solubilize it with a magnetic stirrer (see Note 1).
Sodium hydroxide (NaOH) solution: Dissolve 4 g NaOH pellets in 1 L distilled water, final concentration 0.1 M (see Note 2).
0.1 M Ascorbic acid: 0.15 % Peracetic acid (PAA) (Sigma-Aldrich, cat. no. 77240) (3:1) (v:v) solution: First prepare 0.15 % PAA solution in 1 L distilled water. For this purpose, add 385 μL of PAA stock solution in 1 L distilled water and mix on a magnetic stirrer. Then, add 17.6 g ascorbic acid to the 0.15 % PAA solution and mix with a magnetic stirrer (see Note 3).
0.15 % PAA solution in 70 % ethanol: Dilute 300 mL of 100 % ethanol stock solution in 700 mL distilled water. Later, add 385 μL PAA solution as described in the previous step (see Note 4).
0.15 % PAA solution prepared in 10–15 % hydrogen peroxide (Sigma-Aldrich, cat. no. 216763): First, mix 500 mL H2O2 stock solution with 500 mL distilled water to 10–15 %. Subsequently, add 385 μL PAA in 10–15 % H2O2 solution and mix with a magnetic stirrer (see Note 5).
2.3 Spectrophotometric Determination of DNA Content
Digestion solution: 10 mM Tris–HCl, 50 mM potassium chloride (KCl), 1.5 mM magnesium chloride (MgCl2), 0.5 % Tween-20, and 20 mg/mL proteinase K: add 0.1576 g Tris–HCl, 0.3727 g KCl, 0.0142 g MgCl2, 500 μL Tween-20 to 100 mL distilled water and dissolve on magnetic stirrer. Add 2 g proteinase K to complete the preparation of the stock digestion solution (see Note 6). Prepare right before use.
Phenol:chloroform:isoamyl alcohol: add 2.5 mL phenol, 2 mL chloroform, and 0.5 mL isoamyl alcohol in a total volume of 5 mL (volumetric ratios are 25:24:1) (see Note 7).
3 M Sodium acetate solution: Dissolve 1.2304 g sodium acetate crystals in 5 mL distilled water.
All procedures should be carried out at room temperature unless otherwise specified.
3.1 Isolation of the Bovine Small Intestinal Submucosa Layer
Wash bovine small intestine segments repeatedly with 0.9 % NaCl solution (see Note 8). Carefully remove the adipose tissue around the intestines without tearing the intestine and wash with 0.9 % NaCl solution (see Note 9).
- 2.Cut bovine small intestine segments into 10-cm-long pieces or according to the planned size of the final product; then cut vertically to bring into membrane form (Fig. 1a, b).
Gently remove the outermost layer (Tunica serosa) manually from the small intestine membranes (Fig. 1c).
Remove the muscle layer at the bottom of the intestine (under the serosa layer) in the same way.
Mucosa membrane is the innermost layer of the intestine, which can be removed by washing. Remove the mucosa by serial washing with 0.9 % NaCl solution (see Note 10). At the final stage, separate the SIS layer by mechanically removing the remaining muscle layer (Fig. 1d).
3.2 Decellularization of the Bovine Small Intestinal Submucosa Layer
Wash the separated bovine SIS layers with 0.9 % NaCl solution until the tissue debris is completely removed (see Note 11).
Following the washing procedure, apply two freeze–thaw cycles. For this, store bovine SIS layers at −86 °C (liquid nitrogen is another option) for 15–20 min. Immediately then incubate the specimens at +37 °C until completely thawed (see Note 12).
Upon thawing, wash the bovine SIS layers with 0.9 % NaCl solution at room temperature for about 10–15 min (see Note 11).
Subsequently, treat bovine SIS layers with 0.1 M NaOH solution at room temperature for 1 h (see Note 13).
In order to remove the remaining NaOH solution from the bovine SIS layers, wash the samples with 0.9 % NaCl solution at room temperature (see Note 11).
Incubate bovine SIS layers for 36 h at 37 °C with 0.1 M ascorbic acid and 0.15 % PAA (3:1) (v:v) solution, prepared in distilled water (see Note 14).
Wash again with 0.9 % NaCl solution at room temperature to remove the chemical residues (see Note 15).
Soak bovine SIS layers in 0.15 % M PAA solution prepared in 70 % ethanol for 20–24 h, at room temperature under constant shaking (see Note 16).
Wash again with 0.9 % NaCl at room temperature, to remove any remaining chemical residues (see Note 11).
Thereafter, treat bovine SIS layers with 0.15 % PAA solution prepared in 10–15 % hydrogen peroxide (H2O2) for 12–16 h at room temperature for bleaching and disinfection (see Notes 4 and 5).
At the final stage, repetitively wash bovine SIS layers with 0.9 % NaCl until the chemicals are completely removed. This is followed by lyophilization.
Place decellularized bovine SIS layers in specified molds and freeze at −86 °C for at least 12 h. Then, lyophilize overnight at −80 °C (see Note 17).
Finally, prepare lyophilized bovine SIS layers for sterilization (Fig. 1e, f) (see Note 18).
3.3 Spectrophotometric Evaluation of Decellularization Efficiency
Cut 1 cm2 samples from decellularized and native bovine SIS membranes (see Note 19).
Weigh the samples and record initial weights (see Note 20).
Incubate samples in the digestion solution [10 mM Tris–HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.5 % Tween-20, and 20 mg/mL proteinase K] at 55 °C for 48 h or until the solution is clear, inside a water bath.
After incubation, centrifuge the final solution at +4 °C at 3,000 rpm for 15 min and collect the supernatant.
Add 100 μL mixture of phenol:chloroform:isoamyl alcohol (volume ratio of 25:24:1) onto the collected supernatant. Centrifuge for 15 min at +4 °C and 3,000 rpm (see Note 7).
Add 200 μL of 3 M sodium acetate (pH 5.5) and 500 μL of 95 % ethanol to the mixture, and incubate at −80 °C for 20 min.
Subsequently incubate the samples at 37 °C for 30 min.
Centrifuge the samples at 25 °C and 10,000 rpm for 10 min, and then discard the supernatant.
Prepare the solutions in large volumes for liberal use during the procedure, and store at room temperature until use unless otherwise stated.
The NaOH solution should be prepared in glass conical flask or beaker due to the corrosive nature of NaOH. Wear protective glasses and gloves to avoid skin and eye contact during preparation.
Take note of the PAA stock solution, which is available in varying concentrations (e.g., 30–40 %) in the market. The quantities and volumes are specified for the product code in this protocol. Importantly, PAA stock solution is highly volatile, due to its high amount of acetic acid content, thus handle it under a fume hood and pay attention during pipetting. Add and solubilize ascorbic acid slowly, if high amounts are used. The final solution must be stored at 2–8 °C until use. Prepare the solutions in a glass conical flask or beaker and seal with parafilm for storage.
Perform all steps under a fume hood. If possible, perform in a closed glass bottle under magnetic stirring.
The calculations may change depending on the concentration of H2O2 stock solution used. Commercial H2O2 solutions are available with a range between 1 and 15 %. The present protocol uses 30 % stock solution of H2O2.
Proteinase K solution should be added after the temperature of the main solution reaches 55 °C in the water bath, since proteinase K displays maximum activity at 55 °C. On the other hand, add Tween-20 solution at a later stage and mix at low speeds to prevent foaming during the preparation.
The solution used in this step is corrosive and can cause severe toxic effects upon inhalation or skin contact. Perform all steps under a fume hood. All personnel must wear protective masks.
Following the extraction, small intestine samples should be immediately transferred to the laboratory in cold 0.9 % NaCl solution. The main purpose of the washing step is to clean the inside of the intestine. Do not use any auxiliary equipment at this stage.
The bovine intestine has significant amount of fat tissue, especially in the nodal areas. To avoid rupture of the intestines taken from the ice-cold solution, fat tissue should be eliminated during frozen state.
Standard washing process removes most or entire mucosa layer since it is a thin epithelial structure. Further mechanical separation may be redundant.
At this stage, the use of 0.9 % NaCl solution allows for general tissue cleansing as well as facilitating cell lysis and disruption of DNA–protein interaction through osmotic shock. It is therefore recommended to refresh the salt solution (every 10 min) depending on the size and number of tissue pieces. Perform washing procedure on a shaker.
Perform to disrupt the cell membrane (by the formation of intracellular ice crystals) by the freeze–thaw process. Place the samples on aluminum sheets to freeze at −86 °C. Alternatively, implementation of liquid nitrogen may freeze tissues faster and increase decellularization efficiency.
The alkaline NaOH solution dissolves the cytoplasmic components of the cells and breaks down the nucleic acids. At this stage, the solution may be refreshed depending on the amount of sample. Please refer to Note 3 when working with NaOH solution.
PAA allows the removal of nucleic acid residues, which can increase the effectiveness of the agents used in further steps, while minimally damaging the ECM content. It is important to refresh the solution at least three times depending on the number and size of samples. Check Note 4 while working with PAA.
In addition to Note 11, the washing duration may vary according to the type of chemical to be removed. For this purpose, unlike routine washes, use fresh 0.9 % NaCl solution until pH stabilizes.
The dehydration effect of ethyl alcohol enhances the removal of lipids and cell lysis, besides the PAA effect described in Note 14. The use of ethyl alcohol makes the solution very volatile. Keep the container covered during the decellularization process or work under a fume hood.
Prior to lyophilization, samples must be frozen in membrane form on a flat surface, e.g., aluminum sheet. Avoid stretching the natural SIS layer, since freezing weakens the mechanical properties.
All samples should be sealed in packages before sterilization (gamma, ethylene oxide, etc.). Upon sterilization, samples must be stored at +4 °C until use.
Cut the samples with sterile scissors inside the biosafety cabinet to avoid possible environmental DNA contamination.
It is important that the weights of the tissue pieces are close to each other. Reduce the size of tissue pieces to equalize the weights.
The specified calculation is useful for data acquired by conventional spectrophotometric measurements or with bench-top small-volume spectrophotometers.
Y.M.E. is the founder of Biovalda, Inc. (Ankara, Turkey) and has intellectual properties related to decellularized tissues.
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