preparing cryopreserved rsc96 cells for nanosecond pulsed electric field stimulation

用 nsPEF 刺激的雪旺细胞增强周围神经再生

Each year, millions of people are affected by nerve injuries involving both the peripheral nervous system (PNS) and the central nervous system (CNS)1. Studies have demonstrated that the axonal repair capacity of the CNS is quite limited after nerve injuries, while the PNS shows enhanced capacity due to the significant plasticity of SCs2. Nevertheless, achieving complete regeneration after peripheral nerve injuries remains arduous and continues to pose a significant challenge to human health3,4. Nowadays, autografts have remained a common treatment despite the drawbacks of donor site morbidity and limited availability5. This situation has prompted researchers to explore alternative therapies, including materials6, molecular factors7, and electrical stimulation (ES). As a factor promoting axonal growth and nerve regeneration8, choosing an appropriate method of ES and exploring the relationship between ES and SCs become essential.
SCs are the main glial cells of the PNS, playing a crucial role in the regeneration of the PNS9,10. Following peripheral nerve injuries, SCs undergo rapid activation, extensive reprogramming2,&#160;and transition from a myelin-forming state to a growth-supportive morphology to conduct the regeneration of the nerve2. A substantial proliferation of SCs occurs at the distal end of the injured nerve, while SCs of the distal stump undergo proliferation and elongation to form Bungner&#39;s band, which are necessary to guide axons to grow towards the target organ11. Moreover, SCs from the proximal and distal nerve stumps migrate into the nerve bridge to form SC cords promoting axon regeneration12. Furthermore, previous studies have demonstrated that the synthesis and secretion of relevant factors related to SCs change in cases of peripheral nerve regeneration, including transcriptional factors13, neurotrophic factors14, and myelination regulators13. This also provides indicators for assessing the activity of SCs. Based on these, the promotion of SC proliferation, migration, synthesis, and secretion of relevant factors have been extensively investigated for improving peripheral nerve regeneration15.
Previous studies have demonstrated the possibility of using ES for nerve regeneration1. A widely accepted explanation is that ES can induce depolarization of cell membranes, alter membrane potential, and affect membrane protein functions by changing the charge distributions on these biomolecules1. However, widely applied Intense PEF may cause severe pain, involuntary muscle contractions, and heart fibrillation8. It also increases creatine kinase (CK) activity, decreases muscle strength, and induces the development of delayed onset muscle soreness (DOMS)16. nsPEF is an emerging technique that stimulates test subjects with high-voltage electric fields within a nanosecond pulse duration, and it is gradually being used in cellular-level research17,18. Previous studies have reported that the possible rationale of nsPEF promoting cell proliferation and organelle activity is the formation of membrane nanopores and the activation of ionic channels, which leads to an increase in cytoplasmic Ca2+ concentration19. nsPEF utilizes pulse power technology to charge the cell membrane, producing pulses characterized by short duration, rapid rise time, high power, and low energy density20. These characteristics suggest that nsPEF may be a preferred mode with minimal stimulation side effects8. Furthermore, nsPEF offers advantages such as minimally invasive procedures, reversibility, adjustability, and non-destructiveness to neural tissues compared to surgical interventions. One mainstream research direction of nsPEF in the biomedical field is its application for tumor tissue ablation using high-energy electric field stimulation21,22,23. Some research results indicate that 12-nsPEF can stimulate peripheral nerves without causing damage24. However, at present, there is limited evidence regarding the application of nsPEF in the field of nerve regeneration. Moreover, stimulating SCs using nsPEF is a pioneering attempt, contributing to further in vivo and clinical research. This study explores whether nsPEF stimulation of SCs can promote nerve regeneration and provide a reliable basis for subsequent in-depth and systematic research.

作者聚焦：创新使用 nsPEF 促进周围神经再生

通过纳秒脉冲电场调节雪旺细胞生长以促进体外周围神经再生

Our research focuses on promoting peripheral nerve regeneration by nsPEF stimulated swan cells. We hope to develop a new way to activate swan cells efficiently as nerve regeneration is quite difficult in most cases. We innovatively use a nanosecond post electric field nsPEF to activate swan cells.We are delighted to find that nsPEF may be a preferred mode for peripheral nerve regeneration with minimal stimulation side effects. Besides our protocol provide a reliable basis for subsequent in depth and systematic research. Our laboratory will continually explore the application of nsPEF.In terms of nerve regeneration, we will build up animal models to promote in vivo testing and evaluate the safety of nsPEF in vivo. As there is limited evidence regarding the application of nsPEF in referral nerve regeneration, we hope we can make breakthroughs.

制备冷冻保存的 RSC96 细胞进行纳秒脉冲电场刺激

首先，将装有 1 毫升 RSC96 细胞悬液的冷冻瓶放入 37 摄氏度的水浴中，同时快速摇晃。将细胞悬浮液转移到含有 4 至 6 毫升完全培养基的离心管中，并充分混合。将试管以 1， 000G 离心 3 至 5 分钟，然后丢弃上清液并将细胞重悬于 3 毫升完全培养基中。现在将细胞悬浮液加入含有 628 毫升完全培养基的培养瓶中，并在 37 摄氏度下孵育过夜。一旦细胞密度达到 80% 至 90%，丢弃培养基并用不含钙离子和镁离子的 PBS 冲洗细胞一到两次。然后向培养瓶中加入0.25%胰蛋白酶，0.53毫摩尔EDTA。在孵育结束时，在显微镜下观察细胞脱离。一旦大多数细胞变圆并脱落，请迅速将培养瓶放回工作区域并轻轻敲击。加入 3 至 4 毫升含有 10% FBS 的培养基以停止消化。然后将烧瓶的内容物彻底混合并吸出溶液。离心细胞悬液。加入 1， 000G 五分钟。丢弃上清液后，将细胞重悬于一至两毫升新鲜培养基中，轻轻移液。将细胞悬液转移到新的 T25 烧瓶中。加入 1 比 2 的比例，加入 7 毫升培养基。

preparing-cryopreserved-rsc96-cells-for-nanosecond-pulsed-electric

Research

JoVE Journal

Neuroscience

Preparing Cryopreserved RSC96 Cells for Nanosecond Pulsed Electric Field Stimulation

30 次观看.  Zhejiang University. 首先，将装有 1 毫升 RSC96 细胞悬液的冷冻瓶放入 37 摄氏度的水浴中，同时快速摇晃。将细胞悬浮液转移到含有 4 至 6 毫升完全培养基的离心管中，并充分混合。将试管以 1， 000G 离心 3 至 5 分钟，然后丢弃上清液并将细胞重悬于 3 毫升完全培养基中。现在将细胞悬浮液加入含有 628 毫升完全培养基的培养瓶中，并在 37 摄氏度下孵育过夜。一旦细胞密度达到 80% 至 90%，丢...

视频: 制备冷冻保存的 RSC96 细胞进行纳秒脉冲电场刺激

Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse Electric Field (nsPEF) is an emerging method applicable in nerve electrical stimulation that has been demonstrated to be effective in stimulating cell proliferation and other biological processes. Aiming to assess whether SCs undergo significant changes under nsPEF and help explore the potential for new peripheral nerve regeneration methods, cultured RSC96 cells were subjected to nsPEF stimulation at 5 kV and 10 kV, followed by continued cultivation for 3-4 days. Subsequently, some relevant factors expressed by SCs were assessed to demonstrate the successful stimulation, including the specific marker protein, neurotrophic factor, transcription factor, and myelination regulator. The representative results showed that nsPEF significantly enhanced the proliferation and migration of SCs and the ability to synthesize relevant factors that contribute positively to the regeneration of peripheral nerves. Simultaneously, lower expression of GFAP indicated the benign prognosis of peripheral nerve injuries. All these outcomes show that nsPEF has great potential as an efficient treatment method for peripheral nerve injuries by stimulating SCs.

Here, we present a protocol for applying nanosecond pulse electric field (nsPEF) to stimulate Schwann cells in vitro. The synthesis and secretion ability of relevant factors and cell behavior changes validated the successful stimulation using nsPEF. The study gives a positive view of the peripheral nerve regeneration method.

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse ...