Development of an alternative harvesting method using pH to detach adherent cells from microcarriers

Peripheral nerve injuries are common in Canada, affecting 2.8% of trauma patients treated every year. Current repair strategies are inadequate and repair is often suboptimal with only 25% of patients recovering full motor function and only 3% regaining full sensory function. Because of this, the field is turning toward regenerative medicine to develop a cellular therapy using Schwann cells to repair injured nerves. Schwann cells differentiated from skin derived precursors (SKP-SCs) are a promising cell type as they are easily obtained and allow for autologous therapy. To be able to generate clinically relevant numbers of SKP-SCs, bioreactors need to be used. Since SKP-SCs are an adherent cell type, to be expanded in suspension bioreactors, small spherical beads known as microcarriers need to be used. Our lab has previously shown that these SKP-SCs readily attach to the microcarriers and grow in stirred suspension bioreactors. We have also shown that by controlling the culture parameters, we can increase the maximum cell density compared to conventional static culture methods. One of the biggest hurdles that remains is an efficient harvesting method that can be scaled up to clinical applications. Current cell detachment protocols use enzymatic based solutions to remove the cells from the surface of the microcarriers. These methods work well in removing the cells, however, they are very labour intensive as they require many washing steps and taking the reactors offline. Therefore, we looked into an alternative method for the detachment of SKP-SCs from microcarriers that will allow for an inline detachment process. This new method is based on previous research done in our lab using high pH solutions to dissociate aggregates. First we investigated the detachment efficiency in static. Cells were cultured in 6-well plates until confluency and then harvested with solutions ranging from pH 8-9.5. With a pH of 9 and an incubation time of 30 minutes, we were able to recover 75% of cells when compared to traditional enzymatic harvesting. Following this we performed a qualitative analysis on the detachment of the SKP-SCs from the microcarriers to determine if this method has potential. Small 3mL samples were taken and solutions with pHs 8.5, 9, and 9.5 were added and incubated for 30 minutes and agitated every 5 minutes. We found that the cells detached with a high efficiency after 30 minutes with a pH of only 8.5. This was then quantified while maintaining a viability of above 90%. Following this we tested this method in harvesting full 125mL bioreactors. We evaluated different pH, agitation rates, and incubation times. We also assessed the ability of the cells to reattach to microcarriers and continue to expand over several serial passages to ensure there were no negative effects on the cells. Lastly we looked at using this method in our controlled bioreactors to increase the pH without the addition of anything else. Based on our results, increasing the pH of the culture medium can detach the SKP-SCs from microcarriers at a pH as low as 8.5 which allows for minimal cell damage while still detaching cells. We also noted that when the pH gets too high (\u3e9.5), the microcarriers begin to clump together causing large aggregates of microcarriers which could lead to clogging during the filtration steps. With increasing agitation, higher recovery efficiencies can be achieved indicating that this method of cell detachment has potential for large volume processes

Stem‐cell–based therapies to enhance peripheral nerve regeneration

Peripheral nerve injury remains a major cause of morbidity in trauma patients. Despite advances in microsurgical techniques and improved understanding of nerve regeneration, obtaining satisfactory outcomes after peripheral nerve injury remains a difficult clinical problem. There is a growing body of evidence in preclinical animal studies demonstrating the supportive role of stem cells in peripheral nerve regeneration after injury. The characteristics of both mesoderm‐derived and ectoderm‐derived stem cell types and their role in peripheral nerve regeneration are discussed, specifically focusing on the presentation of both foundational laboratory studies and translational applications. The current state of clinical translation is presented, with an emphasis on both ethical considerations of using stems cells in humans and current governmental regulatory policies. Current advancements in cell‐based therapies represent a promising future with regard to supporting nerve regeneration and achieving significant functional recovery after debilitating nerve injuries.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154610/1/mus26760.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154610/2/mus26760_am.pd

Upper limb nerve transfer surgery in patients with tetraplegia

IMPORTANCE: Cervical spinal cord injury (SCI) causes devastating loss of upper extremity function and independence. Nerve transfers are a promising approach to reanimate upper limbs; however, there remains a paucity of high-quality evidence supporting a clinical benefit for patients with tetraplegia. OBJECTIVE: To evaluate the clinical utility of nerve transfers for reanimation of upper limb function in tetraplegia. DESIGN, SETTING, AND PARTICIPANTS: In this prospective case series, adults with cervical SCI and upper extremity paralysis whose recovery plateaued were enrolled between September 1, 2015, and January 31, 2019. Data analysis was performed from August 2021 to February 2022. INTERVENTIONS: Nerve transfers to reanimate upper extremity motor function with target reinnervation of elbow extension and hand grasp, pinch, and/or release. MAIN OUTCOMES AND MEASURES: The primary outcome was motor strength measured by Medical Research Council (MRC) grades 0 to 5. Secondary outcomes included Sollerman Hand Function Test (SHFT); Michigan Hand Outcome Questionnaire (MHQ); Disabilities of Arm, Shoulder, and Hand (DASH); and 36-Item Short Form Health Survey (SF-36) physical component summary (PCS) and mental component summary (MCS) scores. Outcomes were assessed up to 48 months postoperatively. RESULTS: Twenty-two patients with tetraplegia (median age, 36 years [range, 18-76 years]; 21 male [95%]) underwent 60 nerve transfers on 35 upper limbs at a median time of 21 months (range, 6-142 months) after SCI. At final follow-up, upper limb motor strength improved significantly: median MRC grades were 3 (IQR, 2.5-4; P = .01) for triceps, with 70% of upper limbs gaining an MRC grade of 3 or higher for elbow extension; 4 (IQR, 2-4; P \u3c .001) for finger extensors, with 79% of hands gaining an MRC grade of 3 or higher for finger extension; and 2 (IQR, 1-3; P \u3c .001) for finger flexors, with 52% of hands gaining an MRC grade of 3 or higher for finger flexion. The secondary outcomes of SHFT, MHQ, DASH, and SF36-PCS scores improved beyond the established minimal clinically important difference. Both early (\u3c12 months) and delayed (≥12 months) nerve transfers after SCI achieved comparable motor outcomes. Continual improvement in motor strength was observed in the finger flexors and extensors across the entire duration of follow-up. CONCLUSIONS AND RELEVANCE: In this prospective case series, nerve transfer surgery was associated with improvement of upper limb motor strength and functional independence in patients with tetraplegia. Nerve transfer is a promising intervention feasible in both subacute and chronic SCI

Factors Within the Endoneurial Microenvironment Act to Suppress Tumorigenesis of MPNST

Background: Deciphering avenues to adequately control malignancies in the peripheral nerve will reduce the need for current, largely-ineffective, standards of care which includes the use of invasive, nerve-damaging, resection surgery. By avoiding the need for en bloc resection surgery, the likelihood of retained function or efficient nerve regeneration following the control of tumor growth is greater, which has several implications for long-term health and well-being of cancer survivors. Nerve tumors can arise as malignant peripheral nerve sheath tumors (MPNST) that result in a highly-aggressive form of soft tissue sarcoma. Although the precise cause of MPNST remains unknown, studies suggest that dysregulation of Schwann cells, mediated by the microenvironment, plays a key role in tumor progression. This study aimed to further characterize the role of local microenvironment on tumor progression, with an emphasis on identifying factors within tumor suppressive environments that have potential for therapeutic application.Methods: We created GFP-tagged adult induced tumorigenic Schwann cell lines (iSCs) and transplanted them into various in vivo microenvironments. We used immunohistochemistry to document the response of iSCs and performed proteomics analysis to identify local factors that might modulate divergent iSC behaviors.Results: Following transplant into the skin, spinal cord or epineurial compartment of the nerve, iSCs formed tumors closely resembling MPNST. In contrast, transplantation into the endoneurial compartment of the nerve significantly suppressed iSC proliferation. Proteomics analysis revealed a battery of factors enriched within the endoneurial compartment, of which one growth factor of interest, ciliary neurotrophic factor (CNTF) was capable of preventing iSCs proliferation in vitro.Conclusions: This dataset describes a novel approach for identifying biologically relevant therapeutic targets, such as CNTF, and highlights the complex relationship that tumor cells have with their local microenvironment. This study has significant implications for the development of future therapeutic strategies to fight MPNSTs, and, consequently, improve peripheral nerve regeneration and nerve function

Schwann Cell Delivery Via a Novel 3-D Collagen Matrix Conduit Improves Outcomes in Critical Length Nerve Gap Repairs

INTRODUCTION The current clinical standard for the repair of a long segmental peripheral nerve injury (PNI) requires extensive harvest of nerve autograft. This approach has several limitations including donor morbidity, size mismatch, and duration of surgery. An alternative therapy that employs a guidance channel to bridge long segmental gaps offers several potential advantages, including the ability to add support cells that aid in promoting axonal regeneration. METHODS The authors studied the use of a Neuragen® 3D conduit in combination with autologous SCs (100,000 cells/μl) to promote axonal regeneration across a critical size defect (13 mm) in the sciatic nerve of male Fischer rats. SCs were loaded inside of a 1 cc syringe by applying negative pressure to the chamber with a syringe plumber. Reversed sciatic nerve autografts were used as positive controls and conduits filled with serum only as negative controls (20 animals per group; total of n = 60). Behavioral analysis and electrophysiology assessments were made in vivo. Animals were sacrificed at either 4 or 16 weeks post-injury, dry muscle weights were measured, and grafts underwent immunohistochemical and morphometric analysis. RESULTS SC survival, post-transplantation, was confirmed by the presence of green fluorescence protein (GFP) labeled positive SCs within regenerated fibers. Regeneration and elongation of myelinated axons in all three segments of the graft were significantly enhanced at 16 weeks in the conduit with SCs as compared to the conduit alone and statistically similar to autograft. Nerves repaired with SC-filled conduits exhibited onset latencies and nerve conduction amplitudes similar to contralateral controls and autograft (P < .05). Adding SCs to the conduit also significantly reduced the presence of muscle atrophy as compared to conduit alone (P < .0001). CONCLUSION Repair of long segmental PNI of rat sciatic nerve is significantly enhanced by SC-filled Neuragen® 3D conduits. Significant improvements in total number of myelinated axons, axon diameter and myelin thickness throughout the SC-filled conduits allows for significant improvements in nerve conduction and a subsequent decrease in muscle atrophy

Pseudo-Pseudotumor Cerebri?

A 23-year-old female to male transgender patient (BMI=30) on testosterone therapy presented to the urgent clinic with bilateral disc swelling and preserved optic disc function (Figure 1a-1b). He had a 5 day history of general malaise. Past medical history was pertinent for scoliosis surgery at age 14 in Brazil with an incidental discovery and subsequent resection of a pilocytic astrocytoma

3D printing strategies for peripheral nerve regeneration

After many decades of biomaterials research for peripheral nerve regeneration, a clinical product (the nerve guide), is emerging as a proven alternative for relatively short injury gaps. This review identifies aspects where 3D printing can assist in improving long-distance nerve guide regeneration strategies. These include (1) 3D printing of the customizable nerve guides, (2) fabrication of scaffolds that fill nerve guides, (3) 3D bioprinting of cells within a matrix/bioink into the nerve guide lumen and the (4) establishment of growth factor gradients along the length a nerve guide. The improving resolution of 3D printing technologies will be an important factor for peripheral nerve regeneration, as fascicular-like guiding structures provide one path to improved nerve guidance. The capability of 3D printing to manufacture complex structures from patient data based on existing medical imaging technologies is an exciting aspect that could eventually be applied to treating peripheral nerve injury. Ultimately, the goal of 3D printing in peripheral nerve regeneration is the automated fabrication, potentially customized for the patient, of structures within the nerve guide that significantly outperform the nerve autograft over large gap injuries.</p