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 is shown in Figure 2A. Trimming and segmenting of the nerve were illustrated in Figure 2B–D. After completion of the process, the peripheral nerve maintained its overall morphology without visible structural disruption (Figure 2E).

Histological evaluation of the decellularization is shown in Figure 3. Hematoxylin and eosin (H&E) staining of the native peripheral nerve revealed abundant nuclei, whereas nuclei were largely absent in the decellularized samples (Figure 3A). Quantification of residual DNA, expressed as ng DNA/mg of dry tissue, demonstrated a substantial reduction in DNA content following decellularization (Figure 3B).

Ultrastructural analysis using scanning electron microscopy (SEM) is presented in Figure 4. The fibrous organization of the extracellular matrix (ECM) remained clearly visible after decellularization, indicating preservation of the native ECM architecture (Figure 4).

Assessment of residual detergent and cytocompatibility is shown in Figure 5. Residual SDC levels were minimal following the washing steps (Figure 5A). Cytotoxicity testing showed no significant cytotoxic response in cultured cells exposed to the decellularized peripheral nerve grafts (Figure 5B).

Figure 1: Schematic workflow for the preparation of decellularized human peripheral nerve grafts. Donated human peripheral nerve segments are first prepared by trimming and segmenting to remove surrounding connective tissue. The nerve segments then undergo delipidization using IPA-based solution exchange (Day 1). Sequential decellularization is performed using NaOH (Day 2) and SDC (Day 3) under continuous solution exchange in a peristaltic pump-driven system. The processed grafts are subsequently packaged and sterilized (Day 4) to obtain decellularized peripheral nerve grafts. Abbreviations: IPA = isopropanol; NaOH = sodium hydroxide; SDC = sodium deoxycholate. Please click here to view a larger version of this figure.

Figure 2: Representative images of the peripheral nerve processing workflow. (A) Peristaltic pump-driven solution exchange system used for delipidization, decellularization, and washing. (B) Donated human peripheral nerve before trimming. (C) Peripheral nerve during trimming. (D) Peripheral nerve after trimming and segmenting. (E) Peripheral nerve after completion of the entire decellularization process. Please click here to view a larger version of this figure.

Figure 3: Evaluation of decellularization efficiency. (A) Representative hematoxylin and eosin (H&E)-stained images of native and decellularized human peripheral nerve tissues showing reduced cellular components after decellularization. Scale bars = 100 µm. (B) Quantification of residual DNA content in native and decellularized nerve tissues, expressed as ng DNA/mg of dry tissue. Data are presented as mean ± SD (n = 3). *p < 0.05. Please click here to view a larger version of this figure.

Figure 4: Scanning electron microscopy (SEM) analysis of nerve microarchitecture. Representative SEM images of native (left) and decellularized (right) human peripheral nerve tissues showing preserved extracellular matrix architecture following the decellularization process. Scale bars = 2 µm. Please click here to view a larger version of this figure.

Figure 5: Evaluation of residual detergent and cytotoxicity. (A) Chromatograms of the SDC standard (left) and extracts from decellularized nerve grafts (right) used to assess residual detergent. The red box indicates the region corresponding to the SDC retention time (~3.2 min). (B) Cytotoxicity assessment of extracts from decellularized nerve grafts using L929 fibroblasts following ISO 10993-5 guidelines. Representative images after 48 h incubation show the reagent control, extract from decellularized nerve grafts, negative control, and positive control groups. Scale bars = 50 µm. Please click here to view a larger version of this figure.

Nerve segment length (cm)	IPA treatment (h)	NaOH treatment (h)	SDC treatment (h)
2–3	1.5	6	6
>3–4	2	8	8
>4–5	2.5	10	10

Table 1: Length-specific processing durations for peripheral nerve segments within the range evaluated in this study.

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 the integration of alkaline treatment with reduced detergent exposure. Conventional detergent-based protocols, such as Sondell (3% Triton X-100 and 4% SDC) and the Hudson method (sulfobetaine-10, sulfobetaine-16, and Triton X-200), rely on prolonged, multistep detergent treatments to achieve effective decellularization^9,10. Although these approaches effectively remove cellular components, prolonged detergent exposure may compromise ECM integrity and contribute to residual detergent-related cytotoxicity^19,20,21. In contrast, the present protocol incorporates NaOH to enhance cellular removal while limiting SDC exposure, thereby reducing overall detergent burden and supporting preservation of native ECM architecture.

Another critical aspect of this protocol is the peristaltic pump-driven solution exchange system. Unlike perfusion-based approaches, which can impose internal pressure on the tissue and risk structural disruption²¹, this system enables continuous, controlled solution exchange within a chromatography column. The effectiveness of this approach is supported by multiple analytical outcomes. Histological analysis and quantification of residual DNA confirmed efficient removal of cellular components, while SEM analysis demonstrated preservation of the native ECM architecture. In addition, low residual SDC levels and favorable cytocompatibility results supported the biocompatibility of the resulting graft. Collectively, these findings suggest that the protocol achieves a balance between effective decellularization and structural preservation.

Several technical steps are critical for minimizing procedural variability and maintaining controlled processing conditions. First, careful handling of the nerve segment during trimming, transfer, and washing is essential. Peripheral nerve tissue is mechanically fragile, and excessive compression, folding, or tension can disrupt internal structure and compromise ECM preservation^19,20. Therefore, tissues should be kept moist and handled gently throughout the procedure. Second, uniform solution exchange within the column is a key determinant of process performance. Air bubbles inside the column should be minimized, and the tissue should remain fully immersed and suspended in the solution rather than adhering to the column wall or filter. Tissue adhesion may result in uneven solution exchange, reducing washing efficiency and causing inconsistent exposure to the processing reagents^21,22. Third, the peristaltic pump-driven system must operate stably throughout the procedure. Continuous circulation is required for delipidization, decellularization, and washing; therefore, all tubing connections should be securely fastened to prevent disconnection due to pump-induced pressure or vibration. During sample retrieval, complete drainage of the column should be avoided. Maintaining sufficient liquid volume helps prevent tissue adhesion and facilitates gentle handling during collection.

Nerve segments measuring 2–3 cm were processed with IPA, NaOH, and SDC for 1.5, 6, and 6 h, respectively. Segments measuring >3–4 cm were processed for 2, 8, and 8 h, respectively, whereas segments measuring >4–5 cm were processed for 2.5, 10, and 10 h, respectively. Thus, the treatment durations ranged from 1.5 to 2.5 h for IPA delipidization and from 6 to 10 h for each decellularization step. All other processing parameters, including reagent concentrations, solution volumes, and washing procedures, were maintained consistently across the length groups. Depending on the tubing configuration, the flow rate was set within the specified range and held constant throughout each processing run. The length-specific processing conditions are summarized in Table 1. Adjustments based on nerve diameter, thickness, fascicular structure, or cellular and matrix composition were not systematically evaluated and require further investigation^23,24. For scale-up applications, a multichannel peristaltic pump system may improve throughput; however, consistent flow conditions across channels should be carefully verified to ensure uniform processing.

Several considerations remain for further optimization. Although this protocol substantially reduces overall detergent exposure, SDC is still included, and future refinements may focus on minimizing or replacing detergent use while maintaining decellularization efficiency and ECM preservation^25,26. In addition, peripheral nerve tissue is inherently difficult to standardize due to variation in fascicular structure, diameter, thickness, and cellular and matrix composition^23,24. During large-scale manufacturing, these differences, together with variations in tissue loading and flow conditions, may contribute to batch-to-batch variability. However, donor-to-donor and batch-to-batch reproducibility were not systematically evaluated in the present study. Therefore, further validation using tissues from multiple donors and independent manufacturing batches will be necessary to establish appropriate tissue acceptance criteria, standardize processes, and ensure batch reproducibility.

An additional limitation concerns graft length. Regenerative performance decreases with increasing graft length, and this length-dependent limitation may be more pronounced in acellular nerve allografts than in autografts^3,27. Because acellular nerve allografts lack resident viable Schwann cells, they depend on the migration and proliferation of host Schwann cells to support axonal regeneration²⁷. Saheb-Al-Zamani et al. reported that limited regeneration in long acellular nerve allografts was associated with increased Schwann cell senescence²⁷.

Nevertheless, a previous in vivo study showed that NaOH-based decellularized human nerve grafts supported functional and structural recovery comparable to that of autografts in a 15-mm rabbit sciatic nerve defect²⁸. Contreras et al. also demonstrated axonal regeneration and functional recovery using a decellularized nerve allograft across a 70-mm peroneal nerve defect in sheep²⁹. These studies support the regenerative potential of decellularized nerve grafts, although further evaluation of grafts prepared using the present protocol across clinically relevant graft lengths is required. Autografts are also limited by donor nerve availability and require an additional harvesting procedure, resulting in a second operative site and donor-site morbidity⁴. Acellular nerve allografts may therefore provide a clinically relevant alternative when sufficient autologous tissue is unavailable or when donor nerve harvesting is impractical.

In summary, this protocol provides a practical and adaptable framework for preparing decellularized peripheral nerve grafts. Its emphasis on reduced detergent exposure, controlled solution exchange, and process standardization supports both research applications and potential translational use in peripheral nerve regeneration. Future studies should focus on further development of detergent-free approaches, system scalability, and comprehensive in vivo evaluation of regenerative performance.

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.