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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-cleaved Caspase-3,&#160; (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&#160;</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&#160;</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&#34;&#160;</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&#160;</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&#160;</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&#38;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.

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

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

We are interested in developing a promising regenerative approach to address spinal cord injuries. In this paper, we validate spinal cord organotypic model for testing cellular replacement therapies in spinal cord research. So far, spinal cord organotypic models are maintained in culture for two or three weeks in vitro.And subculture media are suboptimal for neural stem cell engraftment, differentiation, and maturation. Cell replacement therapies still require the improvement to announce the ability of the grafted cells to reconstitute the lost circuits. Through this protocol, we provide a novel, long-term ex vivo platform to tackle cell transplant related issue like survival, integration, and maturation rate of the engrafted neural stem cells.This platform will be helpful for researchers to find the best strategy for cell transplantation, reducing the number of animals required for in vivo validation. Our protocol is simple, fast, and cost-effective to perform proof of concept and optimization studies.

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>

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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.

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

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