Preparation of Decellularized Human Peripheral Nerve Grafts Using Sodium Hydroxide and Sodium Deoxycholate

Summary

This protocol describes a standardized alkaline-assisted decellularization method that combines sodium hydroxide treatment with a peristaltic pump-driven solution-exchange system to produce decellularized human peripheral nerve grafts with effective cellular removal and preservation of the native extracellular matrix architecture.

Abstract

Peripheral nerve injuries often require graft materials that provide structural guidance for axonal regeneration. Although autologous nerve grafting remains the clinical gold standard, its use is limited by donor-site morbidity and restricted tissue availability. These limitations have driven increasing interest in decellularized human peripheral nerve grafts as an alternative biomaterial for nerve regeneration. However, commonly used detergent-based decellularization protocols may disrupt extracellular matrix (ECM) architecture and leave cytotoxic residues that can compromise graft quality. In this article, a standardized protocol is described for preparing decellularized human peripheral nerve grafts using an alkaline-assisted decellularization approach. The procedure begins with sodium hydroxide (NaOH) treatment to promote efficient cellular disruption, followed by a mild sodium deoxycholate (SDC) step that removes remaining cellular and immunogenic components while preserving the native ECM organization. This strategy limits detergent exposure while supporting preservation of the native ECM architecture. The workflow includes donor nerve preparation, controlled NaOH treatment, subsequent mild SDC treatment, and extensive washing under continuous solution exchange using a peristaltic pump-driven system to facilitate removal of residual processing agents. Further post-processing steps are implemented to maintain structural integrity and prepare the grafts for final packaging and sterilization. Decellularization efficiency is assessed through histological analysis and quantification of residual DNA, and the preservation of ECM structure is examined using scanning electron microscopy. The absence of residual SDC and the biocompatibility of processed grafts were confirmed through cytotoxicity testing. This protocol provides a practical approach for producing decellularized human peripheral nerve grafts for peripheral nerve regeneration research and translational applications.

Introduction

Peripheral nerve injuries can lead to severe functional impairment and remain a significant clinical challenge in regenerative medicine and reconstructive surgery. Regeneration of segmental nerve defects requires graft materials that not only bridge the injury gap but also provide structural guidance and biochemical cues to support axonal regeneration and reinnervation. Autologous nerve grafting is widely regarded as the clinical gold standard because it preserves the native extracellular matrix (ECM) architecture and provides a supportive microenvironment for nerve regeneration^1,2. However, its clinical use is limited by the restricted availability of donor tissue, the need for additional surgical procedures, and donor-site morbidity^3,4. These limitations have driven the development of alternative graft materials, particularly decellularized peripheral nerve grafts that retain the native ECM architecture while minimizing immunogenic cellular components^5,6,7,8.

The performance of decellularized nerve grafts is highly dependent on the decellularization process. Conventional methods typically rely on detergent-based reagents such as Triton X-100 and sodium deoxycholate (SDC), which remove cellular components by solubilizing lipid membranes^9,10,11. However, prolonged exposure to these detergents may disrupt ECM microstructure and leave residual cytotoxic substances that negatively affect cellular viability and regenerative outcomes^12,13. As a result, alternative decellularization strategies that enable efficient cell removal while preserving ECM integrity are actively being investigated¹⁴. Alkaline decellularization using sodium hydroxide (NaOH) has emerged as a potential alternative approach because extreme pH conditions can efficiently degrade cellular components, nucleic acids, and lipids^15,16,17. Nevertheless, uncontrolled exposure to strong alkaline conditions may compromise ECM integrity, highlighting the importance of precisely controlled processing conditions^16,18.

Here, a standardized alkaline-assisted decellularization protocol for human peripheral nerve grafts is presented, incorporating a peristaltic pump-driven continuous solution-exchange system. Compared with conventional static immersion methods, this dynamic process enables continuous solution exchange, facilitating the removal of cellular debris and residual processing agents. This article provides a step-by-step visual demonstration of the complete workflow, including donor nerve preparation, controlled alkaline-assisted decellularization, and continuous solution exchange using a peristaltic pump-driven system. The resulting grafts were evaluated using histological analysis, quantification of residual DNA, and scanning electron microscopy (SEM) to confirm efficient decellularization and the preservation of ECM ultrastructure. By enabling clear visualization of critical technical steps, this article provides a clearly defined framework for preparing decellularized nerve grafts and for future process scale-up.

Protocol

Donated human peripheral nerves were obtained from a U.S. tissue bank under an exclusive agreement with L&C BIO and in compliance with the regulations of the American Association of Tissue Banks (AATB) and the U.S. Food and Drug Administration (FDA). Donor information, including age and sex, medical histories, and serological test results, was provided with the tissues and reviewed and approved by a certified medical director. Upon receipt, all tissues underwent incoming inspection, including verification of donor screening, serological testing, and testing for relevant pathogens; only tissues that met the predefined acceptance criteria were used in this study. All procedures involving human specimens were conducted in accordance with the principles of the Declaration of Helsinki. As no human subjects were directly recruited or involved in this study, a separate Institutional Review Board (IRB) approval number was not issued.

1. Prepare the solutions (Day 1)

1. Prepare 0.25 N sodium hydroxide (NaOH) solution by dissolving 10 g of NaOH in 1 L of distilled water under magnetic stirring.
2. Prepare 4% sodium deoxycholate (SDC) solution by dissolving 40 g of SDC in 1 L of distilled water under magnetic stirring.
3. Transfer each prepared solution into sterile glass bottles.
   CAUTION: Handle sodium deoxycholate (SDC) inside a chemical fume hood. Wear appropriate personal protective equipment, including gloves, a laboratory coat, and eye protection, to prevent inhalation and skin contact.

2. Prepare the peripheral nerve segments for decellularization

1. Disinfect the outer surface of the packaged raw peripheral nerve and open the package aseptically.
2. Rinse the peripheral nerve several times with sterile distilled water.
3. Remove residual adipose tissue, connective tissue, unnecessary branches, and damaged nerve tissue using sterile scissors and forceps. Rinse the peripheral nerve again with sterile distilled water.
4. Cut the peripheral nerve into segments measuring 2–5 cm in length. Classify the prepared segments into three length groups: 2–3 cm, >3–4 cm, and >4–5 cm.
   NOTE: Process each length group separately because different IPA, NaOH, and SDC treatment durations are applied according to nerve segment length, as summarized in Table 1.

3. Set up the peristaltic pump system

1. Attach chemically resistant silicone tubing of sufficient length to both ends of the chromatography column.
2. Connect one end of the tubing to the peristaltic pump.
3. Connect the free ends of the tubing to a 1 L glass bottle containing isopropyl alcohol (IPA) using pump tubing to establish a closed-loop circulation system.
   NOTE: Secure all tubing connections firmly, as pressure and vibration from the peristaltic pump may cause the tubing to disconnect from the column or bottle.

4. Delipidize the peripheral nerve

NOTE: Unless otherwise indicated, perform all solution-exchange steps at room temperature (20–25 °C) using a peristaltic pump set to 90–186 mL/min, depending on the tubing configuration. Maintain the selected flow rate constant throughout each processing run.

1. Open one end of the chromatography column and partially fill the column with the IPA solution to ensure that the nerve tissue remains immersed immediately after insertion.
2. Using sterile forceps, gently grasp the outer tissue at one end of a single prepared peripheral nerve segment. Avoid directly compressing or grasping the main body of the nerve. Carefully lower the nerve segment into the solution within the column.
3. Using the forceps, gently straighten the nerve segment and position it longitudinally, parallel to the direction of fluid flow. Ensure that the segment is not folded, coiled, or pressed against the inner wall of the column or the outlet filter.
4. Close the column and position it at a slight upward incline toward the outlet, with the outlet placed slightly higher than the inlet. This orientation helps prevent the nerve segment from settling against either end filter, particularly the outlet filter.
   NOTE: Process one peripheral nerve segment per column per procedure. No additional physical fixation device is required. The natural buoyancy of the nerve tissue and the relatively gentle fluid flow help maintain the segment in a suspended and extended position. Inspect the tissue position during processing and gently reposition it with sterile forceps if it adheres to the column wall, filter, or itself.
5. Connect the IPA solution to the peristaltic pump system. Perform continuous solution exchange through the column for 1.5 h for nerve segments measuring 2–3 cm, 2 h for segments measuring >3–4 cm, or 2.5 h for segments measuring >4–5 cm.
6. Drain the solution from the column.
7. Add 40 mL of sterile distilled water to the column. Gently invert or shake the column five times to wash the peripheral nerve.
8. Repeat steps 4.6 and 4.7 for a total of five washes.
9. Connect a reservoir containing 5 L of sterile distilled water to the peristaltic pump system. Perform continuous solution exchange through the column overnight.
   PAUSE POINT: After completing the overnight wash, the procedure may be paused. Stop the peristaltic pump and store the peripheral nerve fully submerged in sterile distilled water at 4 °C for up to 24 h before proceeding to step 5.1.
   NOTE: Minimize air bubbles inside the column to promote efficient solution exchange. Adjust the peripheral nerve segment as needed to keep it suspended in the solution and prevent it from adhering to the inner wall of the column or filter.

5. Perform the first decellularization (Day 2)

1. Connect the 0.25 N NaOH solution to the peristaltic pump system. Perform continuous solution exchange through the column for 6 h for nerve segments measuring 2–3 cm, 8 h for segments measuring >3–4 cm, or 10 h for segments measuring >4–5 cm.
2. Drain the solution from the column.
3. Wash the peripheral nerve as described in steps 4.6–4.8.
4. Connect a reservoir containing 5 L of sterile distilled water to the peristaltic pump system. Perform continuous solution exchange through the column overnight.
   PAUSE POINT: After completing the overnight wash, the procedure may be paused. Stop the peristaltic pump and store the peripheral nerve fully submerged in sterile distilled water at 4 °C for up to 24 h before proceeding to step 6.1.

6. Perform the second decellularization (Day 3)

1. Connect the 4% (w/v) SDC solution to the peristaltic pump system. Perform continuous solution exchange through the column for 6 h for nerve segments measuring 2–3 cm, 8 h for segments measuring >3–4 cm, or 10 h for segments measuring >4–5 cm.
2. Drain the solution from the column.
3. Wash the peripheral nerve as described in steps 4.6–4.8.
4. Connect a reservoir containing 5 L of sterile distilled water to the peristaltic pump system. Perform continuous solution exchange through the column overnight.
   PAUSE POINT: After completing the overnight wash, the procedure may be paused. Stop the peristaltic pump and store the peripheral nerve fully submerged in sterile distilled water at 4 °C for up to 24 h before proceeding to step 7.1.

7. Sterilize the decellularized peripheral nerve (Day 4)

1. Remove a small volume of distilled water from the column. Carefully retrieve the decellularized peripheral nerve while keeping it suspended in the remaining liquid.
2. Package the peripheral nerve together with sterile distilled water.
3. Sterilize the packaged peripheral nerve by gamma irradiation at a dose of 25 kGy.
   NOTE: Do not completely drain the distilled water from the column. Keep the nerve segment suspended in a sufficient volume of the remaining liquid to prevent it from adhering to the inner wall of the column and to facilitate gentle handling without allowing the liquid to overflow.

Results

An overview of the preparation process for decellularized human peripheral nerve grafts is shown in Figure 1. The workflow included trimming of the raw peripheral nerve, delipidization with IPA, sequential chemical decellularization with NaOH and SDC, followed by extensive washing and sterilization (Figure 1).

The peristaltic pump-driven solution-exchange system used for delipidization, decellularization, and washing ...

Discussion

This study presents a standardized alkaline-assisted decellularization protocol for fabricating decellularized human peripheral nerve grafts for peripheral nerve regeneration. The protocol integrates donor nerve pretreatment, controlled chemical decellularization, and a peristaltic pump-driven solution exchange system into a clearly defined workflow, enabling efficient removal of cellular and immunogenic components while preserving the native ECM architecture.

A key feature of this method is t...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BT100-1L Multi-channel Peristaltic Pump	Longer Pump	BT100-1L	Used to provide continuous circulation and solution exchange during delipidization, decellularization, and washing.
Econo-Column Chromatography Column, 2.5 × 10 cm	Bio-Rad	737-2512	Used to hold peripheral nerve segments during continuous solution exchange.
Isopropyl alcohol (IPA)	Daejeong Chemicals	5035-4410	Used for delipidization of peripheral nerve tissue.
Masterflex Tygon E-LFL Pump Tubing	Thermo Scientific	06440-16	Used to connect the peristaltic pump, chromatography column, and solution reservoirs for continuous solution circulation.
Sodium deoxycholate (SDC)	Sigma-Aldrich	30970-100G	Used as the detergent for the second decellularization step to remove residual cellular components.
Sodium hydroxide, NaOH	Duksan	1636	For cellular disruption.