The study was supported by the Wings for Life Foundation (WFL-IT- 20/21), the European Union Next-Generation EU-National Recovery and Resilience Plan (NRRP)-mission 4 component 2, investment n. 1.4-CUP N. B83C22003930001 (Tuscany Health Ecosystem-THE, Spoke 8), and the Marina Romoli Onlus. This manuscript reflects only the authors&#39; views and opinions, neither the European Union nor the European Commission can be considered responsible for them. Data and metadata are available on Zenodo 10.5281/zenodo.10433147. Images were generated with Biorender https://www.biorender.com/.

Long-Term Engraftment of h-SC-NES Cells in Spinal Cord Organotypic Cultures

The authors have no conflicts of interest to declare.

There is still no effective treatment for patients with SCI. Different approaches have been tested and one of the most promising is based on a regenerative strategy-cell replacement. Currently, the advancements in the regenerative medicine field ask for novel platforms to test the efficacy and safety of cell transplants, alone or in combination with other approaches. Their preclinical validation is essential to pursue further clinical studies. SC organotypic cultures are a useful platform for studying different aspects of neurodegeneration, neural regeneration, and neurodevelopment, and for investigating the effectiveness of novel therapeutic approaches23.&#160;In particular, specific features of the organotypic cultures such as the maintenance of original histoarchitecture and cell and microenvironment composition are advantageous to unravel transplantation dynamics, such as cell engraftment, integration, differentiation, and maturation.
Consistently with published protocols, SC organotypic slices can be maintained in culture for approximately 2-3 weeks in healthy conditions, which limits their use for the long-term investigations and functional screening required for testing schemes of cell therapy. Exploring important processes such as differentiation and maturation towards the correct fate of transplanted cells inside SC tissue requires long-term monitoring. These cellular processes are critical during common transplants in animal models. The availability of an ex vivo system that mimics many features present in vivo would be helpful in the preclinical screening phase.
For this reason, in this work, we propose an optimal long-term (&#8805;30 days) SC organotypic culture method that allows to maintain viable SC slices for up to 90 days, tripling their usual culture time frame. Moreover, we show stable h-SC-NES cell engraftment inside SC slices and the maintenance of the transplant culture for up to 30 days. We monitored cell engraftment over time by observing GFP expression to verify cell survival up to DPT 30. After 30 DPT, we evaluated the cell apoptosis rate. In literature, the evaluation of apoptosis of transplanted h-SC-NES cells in SC-slices at 7 DPT has been reported40. Here, we extended cell apoptosis analysis at DPT 30 to compare the apoptotic rate with respect to the earlier time-point (DPT 7). We found out that our data are in line with the literature, suggesting that transplanted h-SC-NES cells survive also at a later time point if they are maintained in the culture condition optimized in our work. This improved long-term ex vivo platform alone and in the transplant configuration will help researchers in preclinical screening for stem cell-based transplants for SCI. This will allow them to identify the best cell candidate for further in vivo studies promoting the success of the transplants. Moreover, after initial screening, SC organotypic slices could also be used in parallel to the in vivo studies to confirm and corroborate long-term cellular dynamics and behaviors observed in animal models or to support mechanistic studies.
Our protocol describes in detail how to generate this long-term organotypic model, but some critical steps should also be discussed. Concerning the generation of the SC organotypic cultures, there are some challenges during the surgery and the first stages of culture. A well-performed surgery procedure is essential to generate slices that maintain the original histoarchitecture. If the SC is ruined during the isolation, slices can lose their typical anatomic structure and tissue damage can induce an excessive pro-inflammatory insult leading to unhealthy conditions and cell death. The most challenging phase during the surgery is the extraction of the SC from the backbone and the removal of meninges from the isolated SC. The success of these steps depends on the experience of the operator; therefore, a training period before starting with the experiments is recommended.
Coronal sectioning of the SC through a chopper is also a challenging phase. The isolated SC should be placed on the cutting deck exactly perpendicular to the blade. The operator should also place the blade perpendicularly to the cutting deck. These precautions are necessary to ensure the generation of reproducible slices among the same and different experiments. Another important issue is that the time for surgery is limited: the entire slice generation procedure must take ~30 min. If the operator spends more time on surgery and cutting, the SC tissue will suffer and this can impair the success of the culture and the next steps of the experiment.
Once slices are placed on the culture membrane, it is important to feed them correctly. GDNF is necessary to sustain tissue recovery and survival. Cutting with a chopper is traumatic for the tissue and, for this reason, slices are placed soon after the cut into an ice-cold dissection medium to clean away the excess of pro-inflammatory and death-promoting molecules. Then, slices are placed on the culture membranes (cell culture inserts) with fresh medium modified with GDNF to promote a faster recovery and slice adhesion to the membrane. GDNF should be added to the medium every day for the first week in culture because of its short half-life50,51. We observed that slices need the continous presence of GDNF during the first days in culture to promote tissue recovery and viability. In any case, as GDNF presence is important for the entire culturing period, it is strongly discouraged to interrupt GDNF administration at further time points.
During the first week in culture, it is also important to check the slices macroscopically by eye and at the microscope. Translucent tissue and transparency of the borders are signs of proper adhesion of the slices to the membrane and of viable tissue. The necrotic tissue will appear extremely white at first macroscopic sight and the necrotic areas will appear dark grey at the microscope. After some weeks in culture, the morphology of tissue may change: cell movements and tissue adhesion to the membrane can influence this process. We observed, for example, the loss of the central lumen in some slices filled with cells and the loss of dorsal and ventral horn morphology. This happens mainly with smaller slices, while most of them will maintain an anatomic structure close to the original one. Slices are usually generated from the lumbar or thoracic regions because in this way they can have the appropriate size to maintain their original histoarchitecture over time: if they are too small, they lose their architecture while,&#160;&#160;if too big, the central region can undergo into necrosis. Thus, we used the lumbar region of mouse pups to generate slices with the appropriate size for optimal long-term culturing but, in principle, other segments can be considered. Moreover, we opted to use the lumbar region, because ventral and dorsal regions&#160;are more distinguishable from each other. In addition, this region presents tissue areas with a higher percentage of motor neurons and grey matter, which are sites of interest for cell replacement therapies&#160;in SCI.&#160;Concerning transplanting cells into the slices, the main issue is related to the break of the glass microneedle tip. If the hole for the passage of cells is too big, it can cause damage to SC tissue during the microinjection. If it is too small, cell stacking can obstruct the needle, hampering the transplantation process. The transplantation procedure should be completed within 1 h to minimize cell suffering and death.
The proposed protocol provides an optimal and versatile tool for different types of investigations. Here, we apply our long-term platform to validate the transplantation of h-SC-NES cells at the first stages of differentiation inside mouse SC tissue for 30 days. The main novelty of the proposed approach is the optimization of the co-culture protocol. The components of GM sustain long-term neuronal survival of the SC-slices and the transplanted h-SC-NES cells. Indeed, GM, being a serum-free medium, sustains the differentiation of the transplanted cells towards the neuronal fate with respect to the medium previously used for organotypic slice culture47.
Regarding the proposed models for SCI, experiments are usually performed on adult mice. So far, the most important differences between neonatal and adult SC are related to the higher regenerative potential found in neonatal with respect to the adult mice52. However, such differences have no impact on the type of protocol that we are proposing, since here we focus on the response of grafted cells to the hosting tissue environment rather than to the regeneration capabilities of the resident neurons. Another difference between neonatal and adult mice after a&#160;SCI is related to the formation of the glial scar that occurs in adults. This aspect is not taken into account in the proposed model, which does not consider the complex physiopathological processes resulting from primary and secondary injuries.
Regarding the applications, the platform could also be used to investigate the integration between the transplanted cells with resident circuits present in the SC organotypic model. Genetic engineering tools were already used in the CNS to evaluate synaptic connectivity and could be exploited in this regard53,54,55. In particular, the integration could be investigated and validated by assessing the formation of synapses between the engrafted cells and the SC ex vivo tissue.&#160;These long-term organotypic cultures could also be exploited for testing neuroprotective and neuroregenerative agents or novel molecules/materials or to study neurodegenerative disorders that involve the SC. To study specific neurodegenerative disorders, the protocol needs to be adapted for culturing SC slices generated from relevant models, such as transgenic mice carrying specific pathology-associated mutations, at the relevant stage for the pathology (i.e., neonatal, juvenile, adult). In conclusion, our protocol and organotypic cultures in general, being explants of a specific organ, present features that bridge the gap between 2D cell cultures and in vivo models, confirming them as an invaluable tool for both basic research and preclinical testing.

Traumatic spinal cord injury (SCI) has devastating physical, psychological, and economic consequences for patients and caregivers1. Many attempts have been made to promote axonal regeneration in SCI with different approaches2,3,4 and some beneficial effects were demonstrated by the formation of neuronal relays between proximal and distal neurons in the injury site through cell replacement therapies. The interest in cell therapies is still growing5 since transplanted cells can play many roles, including providing trophic support, immune modulation, regenerating lost neural circuits through induction of plasticity, cell replacement, and axon remyelination6.
Recently, the main effort in the field focused on human neural stem/progenitor cells (NSCs/NPCs)7. Several studies suggest that NSCs/NPCs modulate astrocyte response8, promote the secretion of proregenerative factors9,10, and replace missing neuronal cells in SCI11,12. However, studies that support the differentiation of transplanted cells into functional neurons are still poor. Moreover, transplanted cell survival and differentiation in the injured spinal cord (SC) are low13, possibly because transplanted cells take several weeks, even months, to differentiate in vivo. Additionally, current studies have not completely elucidated many biochemical, molecular, cellular, and functional aspects of cell replacement therapies. In this context, simple, fast, and cost-effective models are required to study the mechanisms of cell engraftment, the ability of engrafted cells to proliferate, differentiate into specific types or subpopulations of cells, and form synapses with resident neurons.
Integrating histological studies into electrophysiological recording and transcriptome and proteome profiling is necessary for a full comprehension of the molecular cascade occurring after cell transplantation. This certainly will speed up the design and validation of new cell replacement therapies in preclinical models and clinical studies. Indeed, to date, the use of rodents, large animals, and non-human primates has been worthwhile for elucidating many cellular processes after transplantation14. However, due to the high cost, the high ethical impact, as well as the complexity of the organism, their use is often not straightforward or not adequate to unravel biochemical and molecular processes. In addition, they may present many disadvantages correlated with biological differences, both interspecies (metabolism) and intraspecies variability (sex, age).&#160;&#160;These factors, together with external factors such as stressful situations, could alter the outcome of an experiment and their predictability in terms of therapeutic translation to humans15,16,17.
Thus, many groups employ 2D in vitro cell culture and ex vivo organotypic slices (ex vivo cultures) in addition to animal models. 2D cell culture is the most commonly used system for studying specific biological processes at a single-cell and/or cell population level. Nevertheless, monolayer cell cultures do not reflect the complexity found in a whole organism. The lack of tissue structures and&#160;physiological environment does not allow the 2D culture systems to completely emulate key structural, morphological, and functional aspects of the investigated tissue18,19,20. Organotypic cultures can overcome some of these issues. Organotypic models are based on explanting a fragment of a tissue or organ and maintaining it ex vivo for a limited period21,22. In particular, slices of the explanted tissue are generated with a precise thickness that allows nutrients to easily reach almost all the cells in the slices. They&#160;can be generated from various regions of the central nervous system, such as the hippocampus, hypothalamus, cerebellum, thalamus, cerebral cortex, substantia nigra and striatum, and spinal cord23. Organotypic cultures retain the tissue architecture, the spatial distribution of cells, the cellular diversity, and the environment (i.e., extracellular matrix composition) of the organ of origin. Moreover, they preserve the original neural activity, connections between cells, and in particular, short-distance circuits after the explant.
These aspects provide some advantages for ex vivo cultures with respect to both monolayer cultures and animal models. They retain key tissue features found in vivo but with the reduction of the costs and the possibility to perform different types of molecular, cellular, and functional experiments with an accurate regulation of the culture environmental parameters24,25,26,27,28,29. Organotypic slices can also be exploited to develop models for different neurological disorders by resembling key histopathological features of specific conditions30. Moreover, the retention of the original multicellular tissue environment renders them appropriate platforms for drug screening and for testing neuroprotective and neuro-regenerative molecules and materials.
In this work, we propose the use of SC organotypic cultures as a model to optimize NSC transplants. This is not trivial since optimal culturing conditions are required to guarantee the survival of both the host (SC tissue) and the transplant (NSCs) for weeks. Different research groups engrafted in organotypic cultures, brain-derived and SC-derived, various types of cells. Most of the works showed the transplantation of mesenchymal stem cells31,32,33, olfactory ensheathing cells34, or NSCs35,36,37,38,39,40 and evaluated the interactions of engrafted cells with the host cells, the survival of the whole system, and whether the transplanted cells differentiated into neurons or neuron-like cells inside the ex vivo tissue environment32,33,41. Some of them evaluated the regenerative potential of cells after transplant, observing their axonal growth inside the tissue37,40,41, the myelinating ability of engrafted precursors of oligodendrocytes42, the migration of engrafted cells into the host tissue43, and whether the transplanted cells released factors pushing towards a proregenerative environment31. One limitation of the current studies is that they do not explore the engraftment for a long-term period.
Considering that NSCs seem to require several weeks to differentiate in vivo44,45, this study focuses on how to generate and maintain long-term (&#8805;30 days) mouse SC slices for up to 90 days. Slices were found to retain their original anatomical structure and to maintain a low and stable apoptotic rate over time and high cell viability. We observed diffuse expression of neuronal markers RNA binding fox-1 homolog 3 (RBFOX3) and neurofilament light chain (NFL), with the latter showing an increasing trend of axonal sprouting around the slices over time, attesting to their healthy condition. Moreover, we successfully transplanted into the SC-slices GFP-expressing human SC-derived neuroepithelial stem (h-SC-NES) cells at the first stages of neuronal differentiation. The NSC graft was maintained for 30 days after transplant and cells showed GFP expression for all the period in culture. The apoptotic rate of cells at day post-transplant (DPT) 30 was also found to be in line with respect to the apoptotic rate value observed at DPT 7 in the same cells40. Cells seemed to engraft into the tissue environment and survived up to several weeks.
In summary, our data demonstrate that it is possible to maintain in culture SC organotypic slices for 3 months without compromising their original cytoarchitecture and the tissue environment. Most importantly, they can be exploited to test cell therapies before proceeding with an in vivo experiment, thus reducing the costs and the experimental time. Here, we illustrate in detail all the passages to generate mouse SC organotypic slices and how to maintain them for long-term periods (&#8805;30 days). Moreover, we deeply explain how to perform NPC transplantation into the slices and how to maintain them for downstream analysis.

<table><tbody><tr><td>anti-cleaved Caspase-3,&amp;nbsp; (Asp175) (5A1E) (Rabbit)</td><td>Cell Signaling Technology</td><td>9661S</td><td>1:400</td></tr><tr><td>anti-GFP (Mouse) - monoclonal</td><td>Sigma/Merck</td><td>G6539</td><td>1:400</td></tr><tr><td>anti-Human Nuclei (Mouse) - monoclonal, clone 235-1&amp;nbsp;</td><td>Sigma/Merck</td><td>MAB1281</td><td>1:400</td></tr><tr><td>anti-Human Nuclei (Rabbit)</td><td>NeoBiotechnologies</td><td>RBM5-346-P1</td><td>1:400</td></tr><tr><td>anti-NeuN (RBFOX3) (Rabbit) - polyclonal</td><td>Sigma/Merck</td><td>ABN78</td><td>1:400</td></tr><tr><td>anti-NFL (Mouse)</td><td>Sigma/Merck</td><td>MAB1615</td><td>1:400</td></tr><tr><td>anti-NFL H-Phospho (Rabbit) -polyclonal</td><td>Biologend</td><td>840801</td><td>1:500</td></tr><tr><td>Aqua Polymount</td><td>Poly-sciences</td><td>18606-20</td><td></td></tr><tr><td>B-27</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>BDNF</td><td>Gibco</td><td>PHC7074</td><td></td></tr><tr><td>Blades</td><td>Leica</td><td>118364227</td><td></td></tr><tr><td>Cell culture graded water</td><td>Sigma/Merck</td><td>W3500-500ML</td><td></td></tr><tr><td>Collagen from rat tail</td><td>Sigma/Merck</td><td>C7661</td><td></td></tr><tr><td>Confocal microscope - A1 Confocal Microscope (Eclipse Ti)</td><td>Nikon&amp;nbsp;</td><td></td><td></td></tr><tr><td>D(+)-Glucose</td><td>Sigma/Merck</td><td>G7021</td><td></td></tr><tr><td>Dissecting Forceps</td><td>World Precision Instruments</td><td>15915</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>31330</td><td></td></tr><tr><td>DPBS</td><td>Sigma/Merck</td><td>D8537</td><td></td></tr><tr><td>EGF</td><td>Sigma/Merck</td><td>gf144</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>FGF-2</td><td>Stemgent</td><td>03-0002</td><td></td></tr><tr><td>GDNF</td><td>Sigma/Merck</td><td>SRP3200</td><td></td></tr><tr><td>Glass capillaries, 3.5&quot;&amp;nbsp;</td><td>Drummond Scientific Company</td><td>3-000-203-G/X</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Goat-anti Mouse IgG Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A11029</td><td></td></tr><tr><td>Goat-anti Mouse IgG Alexa Fluor 647</td><td>Thermo Fisher Scientific</td><td>A21236</td><td>1:500</td></tr><tr><td>Goat-anti Rabbit IgG Alexa Fluor 568</td><td>Thermo Fisher Scientific</td><td>A11011</td><td>1:500</td></tr><tr><td>Goat-anti Rabbit IgG Alexa Fluor 647</td><td>Thermo Fisher Scientific</td><td>A21244</td><td>1:500</td></tr><tr><td>Graph Pad-Prism</td><td>Dotmatics</td><td></td><td>Software for Statistical Analysis</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-050</td><td>1:500</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher Scientific</td><td>H3570</td><td></td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>16050-122</td><td></td></tr><tr><td>Insulin</td><td>Sigma/Merck</td><td>I9278</td><td></td></tr><tr><td>Laminin</td><td>Sigma/Merck</td><td>L2020</td><td></td></tr><tr><td>Lentiviral prep</td><td>Addgene</td><td>17446-LV</td><td></td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher Scientific</td><td>25030024</td><td></td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity assay kit</td><td>Thermo Fisher Scientific</td><td>L32250</td><td></td></tr><tr><td>McIlwain Tissue Chopper</td><td>World Precision Instruments</td><td></td><td></td></tr><tr><td>MEM</td><td>Gibco</td><td>11090-081</td><td></td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>5242956003</td><td>to load cells in the needle for transplantation</td></tr><tr><td>Microscope slides</td><td>VWR</td><td>631-0909</td><td></td></tr><tr><td>Millicell cell culture membrane</td><td>Sigma/Merck</td><td>PICM0RG50</td><td></td></tr><tr><td>Miscroscope cover glasses</td><td>VWR&amp;nbsp;</td><td>ECN 631-1572</td><td></td></tr><tr><td>N-2</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Neurobasal</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Petri dish (35mm)</td><td>VWR</td><td>734-2317</td><td></td></tr><tr><td>PFA</td><td>Sigma/Merck</td><td>P6148-500G</td><td></td></tr><tr><td>Plastic pasteur pipette</td><td>Sarstedt</td><td>86.1171.010</td><td></td></tr><tr><td>Pneumatic PicoPump</td><td>World Precision Instruments</td><td>PV830</td><td>Microinjector for transplantation</td></tr><tr><td>Poly-L-lysine</td><td>Sigma/Merck</td><td>P4707</td><td></td></tr><tr><td>Scalpel blade No 10 Sterile Stainless Steel</td><td>VWR International</td><td>SWAN3001</td><td></td></tr><tr><td>Scalpel handle #3</td><td>World Precision Instruments</td><td>500236</td><td></td></tr><tr><td>Spring Scissors</td><td>World Precision Instruments</td><td>501235</td><td></td></tr><tr><td>Stereomicroscope for imaging and acquisition</td><td>Nikon&amp;nbsp;</td><td>SMZ18</td><td></td></tr><tr><td>Stereomicroscope for surgery</td><td>VWR</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Merck</td><td>T8787</td><td></td></tr><tr><td>Tweezers-Dumont #5-inox</td><td>World Precision Instruments</td><td>501985</td><td></td></tr><tr><td>Vannas Scissors, 8.5 cm</td><td>World Precision Instruments</td><td>500086</td><td></td></tr><tr><td>Vertical micropipette puller</td><td>Shutter Instrument</td><td>P-30</td><td></td></tr><tr><td>Y-27632</td><td>R&amp;amp;D Systems</td><td>1254/50</td><td></td></tr></tbody></table>

long-term mouse spinal cord organotypic slice culture as a platform for validating cell transplantation in spinal cord injury

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs.
Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (&#8805;30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

The described methods allow the establishment of SC organotypic slices from mice at stage P3 and their maintenance in culture for a prolonged time in healthy conditions. Moreover, we show a protocol for transplanting cells into the slices and for co-culturing them for up to 30 days (Figure 1). First, we show the optimization of the culture conditions and a protocol suitable for prolonged culturing of the SC slices with transplanted cells (Figure 2A). Slices are generated and maintained from DEV 0 until DEV 2 in the OM, which was originally proposed as an optimal medium for the maintenance of SC slices47. However, due to the presence of serum proteins, this medium could be suboptimal to sustain the neuronal differentiation and maturation of the transplanted neural precursor cells. Indeed, at DEV 3, we tested the switch from OM to the GM, a formulation containing Neurobasal plus B27, which supports neural survival, and without serum, which inhibits the correct neuronal differentiation, promoting instead a glial fate48,49.
Figure 2B shows the results achieved by switching the medium at DEV 3 from OM to GM, compared to the SC slices not receiving the switch (control slices were cultured in OM). We used the distribution of the NFL signal inside the slices as a marker for neuronal integrity (Figure 2B,C). Slices at DEV 7 were healthy in both culturing conditions, showing the diffuse distribution of neurofilament (NFL, in green) inside them. At DEV 10, slices cultured in GM seemed to be healthier with respect to the control slices cultured in OM, as documented by NFL staining distribution. We also estimated the NFL+ area (% NFL+ Area/DAPI+ Area) of the slices shown in the representative images of Figure 2B. The estimated NFL+ area is represented in the histograms in Figure&#160;2C, confirming that the NFL signal is diffusely distributed in the slices at DEV 7 under both conditions. However, at DEV 10, the estimated area covered by NFL staining decreases for the OM culturing condition.
These data suggest that switching to the GM at DEV 3 is well-tolerated for prolonged culturing of SC slices (DEV 10). As a next step, we tested GM at more prolonged time points: DEV 30, DEV 60, and DEV 90. As shown in Figure 3A,B, slices were maintained healthy in culture until DEV 90. NFL staining was found widely present in the slices at each time point, with diffuse sprouting around the slices of neurites departing from the central region. Indeed, we estimated the NFL+ area of the slices shown in Figure 3A and it increased over time as shown in the histograms of Figure&#160;3B. We also observed positivity to the neuronal marker RBFOX3, providing another line of evidence of the neuronal differentiation of the slices. At each time point, we also checked the apoptosis rate by evaluating in different slices the number of cells positive to aCASP3 (Figure 4A,B). The analysis was performed as described in protocol section 10.2. Apoptotic rate (% aCASP3+ cells/total number of DAPI+ cells) was found to be very low at each time point (0.85 &#177; 0.52%, 0.71 &#177; 0.27%, 0.66 &#177; 0.45% for DEV 30, 60, and 90, respectively) with no significant differences between the three considered time points (p-value &#62; 0.05, Figure 4B). These data suggest that the apoptotic rate associated with aCASP3 remains stable during time and, together with the wide distribution of NFL in the slices (Figure 4A), confirm&#160;the survival of the slices at each time point.
In support of previous data, we also performed a live/dead assay to evaluate the viability of the slices at the three different time points. We used Calcein (green staining) to label the viable and metabolically active cells and Sytox (cyan staining) to assess cell death. As shown in the histograms in Figure 4C, the percentage of metabolically active cells increases slightly from DEV 30 to DEV 90 (93.17 &#177; 5.21%, 96.43 &#177; 3.02%, 96.33 &#177; 3.10% for DEV 30, 60, and 90, respectively), stabilizing between the last two time points (DEV 30 vs DEV 60 p-value = 0.018; DEV 30 vs DEV 90 p-value = 0.027; DEV 60 vs DEV 90 p-value = 0.99). We found low levels of cell death that decreased over time (6.83 &#177; 5.21%, 3.57 &#177; 3.02%, 3.66 &#177; 3.10% for DEV 30, 60, and 90, respectively) and a significant difference was found between DEV 30 and later time points, DEV 60 and DEV 90 (DEV 30 vs DEV 60 p-value = 0.018; DEV 30 vs DEV 90 p-value = 0.027; DEV 60 vs DEV 90 p-value = 0.99). These data, in association with the apoptosis rate, confirm slice survival over time and support the effectiveness of the long-term culturing protocol performed.
Once the feasibility of prolonged culturing of the SC slices was established, we challenged the system by transplantation of h-SC-NES cells at the first stages of neuronal differentiation. We tested the h-SC-NES cells because they have shown promising results for SCI treatment12. The transplantation procedure of h-SC-NES cells into the mouse SC slices is described in protocol section 6. The SC slices and transplanted h-SC-NES cells were maintained until DPT 30. Cells were grafted at DIV 10 of differentiation (neural precursor stage) into DEV 4 organotypic slices, as shown in the protocol scheme of Figure 5A. Transplanted cells were monitored for the expression of GFP in culture for up to 30 days. Figure 5B shows representative live images, at different DPT, of an SC slice with transplanted GFP+ cells. The stable expression of GFP over time (Figure 5B and Figure 6A) suggests that cells survived into the SC tissue in the previously optimized culture conditions. We also checked the apoptotic rate of transplanted cells as described in protocol section 10.2. The apoptotic rate (% aCASP3+ cells/total number of Hu-Nu+ cells) was found to be very low (0.44 &#177; 0.34%) after 30 DPT (Figure 6B). Moreover, the apoptotic rate at DPT 30 was found to be in line with that found for the same type of cells at DPT 7, as previously reported40, documenting that the cultures stabilize over&#160;time.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66704/66704fig01.jpg" /> 
Figure 1: Workflow of the protocol. Representative scheme showing the general workflow of the protocol performed. (A) On the left, a scheme summarizing&#160;mouse SC-slice generation from isolated SC of mouse pups at P3 and long-term culturing of SC organotypic slices. (B) On the right, a scheme summarizing the transplantation of h-SC-NES cells expressing GFP into mouse SC-organotypic slices. Grafted cells are maintained for 30 days post-transplant. Abbreviations: h-SC-NES = human spinal cord-derived neuroepithelial stem; GFP = green fluorescent protein; DEV = day&#160;ex vivo; DPT = day&#160;post-transplant; NFL = neurofilament light chain; RBFOX3= RNA binding fox-1 homolog 3; aCASP3 = active Caspase-3; SC= spinal cord. <a href="https://www.jove.com/files/ftp_upload/66704/66704fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66704/66704fig02v2.jpg" /> 
Figure 2: Optimization of long-term culturing conditions. (A) Representative scheme of the protocol for testing OM and GM. OM is maintained until DEV 7-10 for the control group. The medium is switched to the GM at DEV 3 for the treated slices; then, they are fixed at DEV 7-10 for comparison to controls. (B)&#160;Representative images comparing mouse SC organotypic slices at DEV 7 and 10 cultured in different conditions. Slices are stained for the cytoskeletal marker neurofilament (NFL, green). The wide distribution of NFL staining in slices cultured with GM suggests an overall survival and differentiation.&#160;Nuclei are counterstained with DAPI. Scale bar = 500 &#181;m. (C)&#160;Representative histograms of the estimate of the area covered by NFL in the slices shown in Figure 1B. At DEV 10, NFL surface area decreases in the OM culturing condition.&#160;Abbreviations: DEV = day&#160;ex vivo; DAPI = 4&#39;,6-diamidino-2-phenylindole; NFL = neurofilament light chain.; OM = organotypic medium; GM = graft medium; SC = spinal cord. <a href="https://www.jove.com/files/ftp_upload/66704/66704fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66704/66704fig03v3.jpg" /> 
Figure 3: Long-term cultured mouse SC organotypic slices. (A) Slices are maintained in culture until DEV 90. Immunofluorescence assay reveals a wide distribution of the cytoskeletal marker neurofilament (NFL, green) and the nuclear neuronal marker RBFOX3 (red), attesting to their healthy condition and neuronal identity after long-term culturing. Of note, NFL+ axons sprout out diffusely around the slices over time. Nuclei are counterstained with DAPI. Scale bar = 500 &#181;m. (B)&#160;Representative histograms of the estimate of NFL+ area and time and, RBFOX3+ area of the slices shown in panel A. NFL+ neurite area increases over time. Abbreviations:&#160;DEV = day&#160;ex vivo; DAPI = 4&#39;,6-diamidino-2-phenylindole; NFL = neurofilament light chain; SC = spinal cord; RBFOX3= RNA binding fox-1 homolog 3. <a href="https://www.jove.com/files/ftp_upload/66704/66704fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66704/66704fig04.jpg" /> 
Figure 4: Evaluation of cell viability in the SC slices over time. (A) Representative images of organotypic slices at DEV 60 stained for aCASP3 (red) and NFL (green). Scale Bar = 100 &#181;m. NFL shows a diffuse pattern. Rare cells are positive for the apoptotic marker aCASP3 (Insets: 1-2-3). (B) Analysis of the apoptosis rate in slices at different time points. Mean &#177; SD, N (replicates) = 6 slices, n (total cells) &#62; 1,000 for each slice, Kruskal-Wallis test, multiple comparison, p-value &#62; 0.05. The apoptotic rate is stable over time. In the insets 1-2-3 of panel A, it is possible to observe details of cells positive for aCASP3 (red staining, white arrows). Small red dots label cell debris and pyknotic nuclei. Scale bar = 50 &#181;m. (C) Representative images of live/dead assay performed on SC slices at DEV 90: metabolically active cells are labeled in green with Calcein, while dead and damaged cells are labeled in light blue (cyan) with Sytox. The two histograms show the % of cells positive for Calcein (on the left) and Sytox (on the right) on the total number of cells. For both mean &#177; SD, N (replicates) = 6 slices, n (total cells) &#62; 1,000 for each slice, Kruskal-Wallis test, multiple comparison, DEV 30 vs DEV 60 p-value = 0.018; DEV 30 vs DEV 90 p-value = 0.027; DEV 60 vs DEV 90 p-value &#62; 0.99. Abbreviations:&#160;DEV = day&#160;ex vivo; DAPI = 4&#39;,6-diamidino-2-phenylindole; NFL = neurofilament light chain; SC = spinal cord; aCASP3 = active caspase-3. <a href="https://www.jove.com/files/ftp_upload/66704/66704fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66704/66704fig05.jpg" /> 
Figure 5: h-SC-NES cell transplantation into mouse organotypic slices. (A) Representative scheme of the transplantation protocol. Cells are transplanted as neural precursors at DIV 10 of differentiation into DEV 4 organotypic slices. (B)&#160;Representative images of mouse organotypic slices transplanted with GFP-expressing h-SC-NES cells over time until DPT 30. Cells are transduced with a lentiviral vector carrying the GFP gene. GFP expression over time confirms their viability and adaptation to the slice environment. Scale bar = 500 &#181;m. Abbreviations: DIV = first day in pre-differentiation; h-SC-NES = human spinal cord-derived neuroepithelial stem; GFP = green fluorescent protein; DEV = day&#160;ex vivo; OM = organotypic medium; GM = graft medium; DPT = days post-transplant. <a href="https://www.jove.com/files/ftp_upload/66704/66704fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66704/66704fig06.jpg" /> 
Figure 6: Apoptosis rate evaluation of transplanted h-SC-NES cells after 30 days from transplant. (A) Representative image of a mouse organotypic slice transplanted with GFP-expressing h-SC-NES cells. Cells are transduced with a lentiviral vector carrying the GFP gene for monitoring them into the slices after transplantation. GFP expression over time confirms their viability and adaptation to the slice environment. The time point shown is DPT 30; cells are stained for human nuclei (cyan) and aCASP3 (red). Scale Bar = 150 &#181;m. (B) On the left, representative pie chart of the apoptosis analysis of cells transplanted in slices at DPT 30 (N (replicates) = 5 slices, n (cells) = 5,000), and on the right, an inset of Hu-Nu+ cells and a detail of a cell positive to aCASP3 (white arrow). Scale bar = 75 &#181;m. Small red dots label cell debris and pyknotic nuclei. Abbreviations: h-SC-NES = human spinal cord-derived neuroepithelial stem; GFP = green fluorescent protein; DPT = day&#160;post-transplant; DAPI = 4&#39;,6-diamidino-2-phenylindole; NFL = neurofilament light chain; aCASP3 = active caspase-3; Hu-Nu = human nuclei. <a href="https://www.jove.com/files/ftp_upload/66704/66704fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of solutions used in this protocol. <a href="https://www.jove.com/files/ftp_upload/66704/66704_Tables of Solutions rev.xlsx" target="_blank">Please click here to download this Table.</a>

Read this Scientific Article Protocol about Author Spotlight: Long-Term Spinal Cord Slice Culture for Advancing Spinal Cord Regeneration Therapies at JoVE.com

Author Spotlight: Long-Term Spinal Cord Slice Culture for Advancing Spinal Cord Regeneration Therapies

In this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches ...

long-term-mouse-spinal-cord-organotypic-slice-culture-as-platform-for

Research

JoVE Journal

Neuroscience

1.4K Views.  University of Pisa. In this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies. 

Video: Author Spotlight: Long-Term Spinal Cord Slice Culture for Advancing Spinal Cord Regeneration Therapies

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Bioengineering

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

Medicine

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...