Modern cell culture technologies: Revolutionizing neuroregeneration in neuropsychiatry

INTRODUCTION

Neuropsychiatry is a medical field that includes both neurology and psychiatric disorders.^[1] Research on neuroregeneration has revolutionized treatments for many of the ailments in this sector. The dynamic discipline of neuroregeneration focuses on the development, maintenance, and connectivity of neurons. In adults, neuronal cells exhibit limited regenerative capabilities. Consequently, severe peripheral nerve injuries often remain unresolved, culminating in paralysis. The placement of a nerve graft is typically necessitated for the restoration of function. Neurogenesis, neuroplasticity, and neurorestoration are three areas of research that show promise for enhancing brain health and curing neuropsychiatric disorders.^[2]

This study aims to provide a concise overview of the uses of contemporary cell culture technologies, including microfluidics, organoids, coculture systems, three-dimensional (3D) culture, and nanofiber scaffolds, in neurodegenerative therapy for major neuropsychiatric disorders. Thanks to these state-of-the-art methods, research on neuroregeneration has progressed significantly, as human brain shape and function can now be better modeled in vitro. We examine current research that models neuropsychiatric illnesses and creates novel treatment strategies using these technologies.

In neuropsychiatry and neuroregeneration research, cell culture techniques are widely employed to investigate and develop treatments for a range of neurological diseases. For the purpose of study and drug development, scientists can cultivate and modify brain cells through the use of cell culture. Cell culture research can help find shared pathways across diverse diseases and potential therapy targets for neuronal preservation and repair, which is beneficial for neurodegenerative disorders, which are common in neuropsychiatry.^[3,4]

D CULTURE

The intricacy of the human nervous system and the limited accessibility of living brain tissue represent considerable hurdles in the research of nervous system and brain development.^[5] To address these issues, 3D cultures [Figure 1] have emerged as a viable way to investigate human brain function. Unlike two-dimensional systems, which are incapable of capturing the delicate interactions between neural cells, 3D neuronal cultures have shown the ability to duplicate human neuron properties such as transcriptional patterns^[6] and in vivo-like neuron-neuron interactions,^[7] including the extracellular matrix (ECM) and its interactions with neurons.^[8] These 3D models expand beyond neurons to incorporate glial cells,^[9] allowing for a better understanding of their interactions with neurons^[5] and reproduction of oligodendrocyte function.^[10] Notably, 3D brain organoids with induced pluripotent stem cell (iPSC)-derived human microglia-like cells that mimic their functional features and transcriptomic profile have been generated.^[11]

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Figure 1:

3D cell culture.

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ORGANOIDS MODELLING NEUROLOGICAL DISEASES

Researchers have created in vitro neurological organogenesis models employing iPSCs to grow human cerebral organoids. These organoids closely resemble the human brain’s multilayered structure [Figure 2] and numerous cell types, which include astrocytes, oligodendrocytes, neuroepithelial cells, neuronal cells, and radial glial cells. They can even reproduce specific layers inside the cortical plate and be used to build different brain areas, allowing researchers to examine and imitate neurological illnesses. These cerebral organoids’ neurons have synapse structures and respond to stimuli with calcium action potentials.

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Figure 2:

Brain organoids.

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Furthermore, these cerebral organoids can be made up of a variety of cell types, including neural stem cells (NSCs), mature and immature neurons, and glial cells, promoting neuronal connections comparable to that found in the human brain in vivo. This enables the investigation of structural phenotypes as well as a better knowledge of the cellular and molecular mechanisms behind human brain development, disease-induced neuronal abnormalities, and cognitive deficits.^[12]

Aside from typical organoids, region-specific spheroids have been used to examine cell migration and brain circuit creation in formations^[13] known as “assembloids.” These organoids can be utilized to study neuronal connections in various brain regions. Furthermore, organoids maintained at the air-liquid interface have improved neuronal survival and maturation, allowing for the formation of long, dense bundles of axons with precise orientations.^[14]

Recent advances in the field have resulted in the production of microglia-containing human brain organoids, which mimic essential aspects of the human brain and open up new avenues for research into brain development and pathology.^[15] These organoids are useful for studying the underlying causes of neurological illnesses because they may develop intricate brain networks and exhibit spontaneous electrical activity.

COCULTURE

Coculturing [Figure 3] is a novel technique in neuroregeneration research that is propelling the field forward. Barberio et al. study, for example, produced a unique coculturing system comprising endothelial cells, astrocyte cells, and neuronal cells. This method allows for the investigation of the neurovascular unit’s barrier functions, which is critical for understanding drug distribution to the central nervous system (CNS). Because many medications have difficulty crossing the blood-brain barrier (BBB), this coculturing strategy provides a platform for future drug testing and delivery pathways, potentially resolving this issue.^[16]

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Figure 3:

Coculture of neurons, microglia, and astrocytes.

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There is also a study on the development of human iPSCs (hiPSCs) into microglia-like cells through specific factor exposure and coculturing with astrocytes. These iPSC-derived microglia (iPS-MG) have the phenotypic, gene expression profile, and functional features of brain microglia. In treating mouse syngeneic intracranial malignant gliomas, murine iPS-MG produced using a similar approach displayed equivalent efficacy to primary brain-isolated microglia. This breakthrough opens up new avenues for research into human microglia and their potential uses in personalized medicine.^[17]

MICROFLUIDICS

Microfluidics-based cell culture platforms [Figure 4] are game-changing drug screening and development technologies, seamlessly merging ideas from biology, biochemistry, engineering, and physics. They use microfluidics, which includes precise manipulation of small liquid volumes in microscale channels, to establish controlled settings for cell culture. These platforms enable 3D cell culture by simulating native tissue conditions. This is critical for understanding cell behavior in physiologically relevant situations. Microfluidic devices enable fine control of fluid flow, concentration gradients, and cell configuration, resulting in precise experiments. Many microfluidic systems are automated, which allows numerous experiments to run at the same time, saving time, and resources.

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Figure 4:

Microfluidics-based cell culture platform.

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Cell culture systems based on microfluidics are used in a variety of sectors, including drug discovery, tissue engineering, and biological research. They make it easier to investigate cell activities and reactions to stimuli. These platforms allow for real-time observation of cell activities, which aids in dynamic studies. Microfluidic systems frequently save money since they require fewer reagent and cell quantities. They enable investigations using fewer cells and animals, which aligns with ethical concerns about animal testing and cell procurement.^[18]

NANOFIBER SCAFFOLDS

There has been a tremendous increase in interest in electrospun nanomaterials derived from cellulose and its derivatives during the past two decades. These materials have attracted interest for a variety of clinical applications, with a particular emphasis on their use in cell culture for tissue engineering. The exceptional mechanical resilience of cellulose filaments is due to their hierarchical structure and the varying presence of crystalline domains. This extraordinary stability is critical in the creation of materials for tissue engineering, ensuring that these structures can efficiently assist cell proliferation and tissue synthesis.

Cellulose and its derivatives have been shown to be biocompatible with a wide range of cell lines. This means they are well tolerated by cells and cause no harmful effects. This biocompatibility is important when creating materials for cell culture since it ensures that the cells can survive in their surroundings. These materials have led the way for the development of polymeric matrices with nanofibrous ECM-like properties. These matrices are useful for tissue engineering applications because they imitate the natural environment that cells experience within tissues and organs. Within these matrices, cells can attach, develop, and interact, promoting the formation of functional tissues [Figure 5].^[19]

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Figure 5:

Nanofiber cell culture platform.

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We present the latest reports at the interface of these modern cell culture technologies and specific neuropsychiatric disorders [Table 1].^[20,21]

ALZHEIMER’S DISEASE (AD)

AD is a type of dementia characterized by gradual loss of mental faculties such as memory, attention, calculation, and judgment. The AD neuropathological signatures include amyloid beta-plaques and neurofibrillary tangles (NFT), leading to neurodegeneration.^[22] With increasing prevalence across the globe, neurogenerative therapies are the hope for this debilitating disease.

STROKE

In medical terminology, “stroke” refers to a wide range of disorders involving the blockage and/or hemorrhaging of blood vessels. The restriction and blockage invariably result in a lack of blood supply and interrupted blood flow. This causes devastating brain deficits, which can sometimes be irreversible. Novel cell culture technologies are being used to try to find treatments for this crippling disease.^[28]

TRAUMATIC BRAIN INJURIES (TBIS)

TBI is described as short- or long-term brain damage caused by external mechanical forces such as accelerating, decelerating, and spinning forces that cause direct physical disturbance of neural tissues.^[33] At present, there is no clinically validated therapy for repairing cerebral parenchyma loss caused by TBI. As a result, neurogenerative technologies are urgently needed to address this problem.

SPINAL CORD INJURY (SCI)

SCI produces terrible paralysis, which may end in the patient being permanently bedridden and imposing a lifelong physical and mental strain on the patient’s family. The neurological condition is characterized by primary mechanical injury and secondary inflammatory response-mediated harm, neither of which has an effective treatment.^[38]

PERIPHERAL NERVE DISEASE

In adults, neuronal cells exhibit limited regenerative capabilities. Consequently, severe peripheral nerve injuries often remain unresolved, culminating in paralysis. The placement of a nerve graft is typically necessitated for the restoration of function. Modern cell culture methods offer novel avenues that could revolutionize the treatment of even severe peripheral nerve injuries by neuroregeneration.

BIPOLAR DISORDER

In the treatment of depression, stem cell therapy has shown potential. Neurogenesis plays an important role in depression, as decreased neurogenesis in the hippocampus is frequently reported in depressed individuals, potentially leading to depressive symptoms. On the other hand, increased neurogenesis has the potential to mediate antidepressant effects.^[44]

Zhang et al. performed a study in which they used HUC-MSCs to treat depression-like symptoms associated with myocardial infarction. They discovered that infusing HUC-MSCs dramatically enhanced cardiac function and depression-like behavior by regulating microglial polarization through Jmjd3 gene downregulation and microglia polarization.^[45]

Furthermore, evidence indicates that glutamatergic anomalies and glial pathology, as well as monoaminergic system malfunction, play an important role in depression. Mesenchymal stem cells expressing glutamate transporters, for example, might restore normal glutamatergic transmission and brain-derived neurotrophic factor levels, potentially reducing depression symptoms.^[46]

Furthermore, increased proinflammatory cytokine release, such as monocyte chemoattractant protein-1, interleukin-1, interleukin-6, and tumor necrosis factor-alpha, might result in depression-like behaviors. Therapy using adipose-derived mesenchymal stem cells has been found to reduce these cytokines and alleviate depression symptoms.^[47]

Overall, stem cell therapy has the potential to modulate depressive illnesses through mechanisms such as neurogenesis, glutamatergic transmission modulation, and anti-inflammatory effects. These findings suggest possible therapeutic implications in the treatment of depression.^[48]

AUTISM

Recent research has investigated the use of cell-based techniques to treat autism spectrum disorders (ASD). One study focused on fetal stem cell (FSC) transplants in autistic children. Pre-transplantation, six months, and 12 months after the procedure, the participants were thoroughly observed. FSCs were injected both intravenously and subcutaneously. Autism Treatment Evaluation Checklist and Aberrant Behavior Checklist assessments were used. The findings revealed no substantial adverse events, such as infections or immunological problems. When compared to pre-treatment values, statistically significant improvements were detected in a variety of domains, including speech, sociability, sensory perception, and general health, as well as reductions in total scores.^[49]

Another study looked at the use of mesenchymal stromal cells (MSCs) to treat neuroinflammation in children with ASD. Children were given intravenous infusions of human cord tissue mesenchymal stromal cells (hCT-MSCs) in this open-label phase I research. Except for a few episodes of agitation during administration, these therapies were well-tolerated. Some youngsters produced anti-human leukocyte antigen (HLA) antibodies, but they had no clinical consequences. Importantly, six of the 12 participants improved on ASD-specific assessments.^[50]

Furthermore, some studies have revealed that hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) may help alleviate ASD symptoms by lowering inflammation and boosting stem cell mobilization, proliferation, and differentiation in tissues. These findings point to promising improvements in the use of cell-based treatments to treat ASD, with an emphasis on safety and efficacy.^[51]

SCHIZOPHRENIA

A recent study concluded that a single intracerebroventricular injection of bone marrow-derived MSC can suffice for long-term reversal of changes in adult hippocampal neurogenesis and improve schizophrenia-like behavioral phenotype inflicted by developmental exposure to ketamine in mice.^[52]

OTHER TECHNOLOGIES

1. Dental pulp stem cells (DPSCs): At present, the transplanted DPSCs alone or in combination with several novel nerve conduits, such as ethyl acrylate collage, are being used in 3D culture. Several current studies have successfully applied DPSCs in nerve regeneration.^[53] In an in vivo rat sciatic nerve injury, nerve conduit preloaded with DPSCs promoted functional repair.^[54] Hypoxia-treated DPSCs transplantation into damaged rat spinal cord increased vascularization and oxygenation of the injured spinal cord.^[55] Pre-differentiated human DPSCs (hDPSCs) were injected into the striated urethral sphincter and transplanted into the pudendal nerve of female rats whose pudendal nerves had been surgically severed to cause stress urinary incontinence. After four weeks, the external urethral sphincter’s thickness significantly recovered as a result of the hDPSCs’ effective engraftment. These transplanted stem cells also showed in vivo loyalty to the myogenic lineage. It is significant to note that the hDPSCs supported vascularization in the afflicted area. There was a discernible increase in incontinence as a result of these cellular treatments, emphasizing the regenerative potential of hDPSCs in treating stress urinary incontinence.^[56] In a DPSC auto-transplantation in the unilateral hindlimb of diabetic rats, in vivo in diabetic rat sciatic nerve injury, improved blood flow, nerve conduction velocity, capillary number, and intradermal nerve fiber density.^[57] The results of a recent study suggested that dental follicular cells could be a viable source of dopaminergic neurons for functional transplantation, and they also prompted further thorough research on the subject’s potential as a PD treatment.^[58] Mead et al.^[59] conducted retinal damage studies and discovered substantial results. They discovered that DPSCs produce many neurofibrillary tangles (NTFs). This NTF release resulted in increased proliferation of neural III-tubulin+ retinal cells and prolonged neuritis. Furthermore, transplanting DPSCs into the vitreous fluid of mice following optic nerve injury enhanced the survival of Brn-3a+ retinal ganglion cells and encouraged axonal regeneration. About 44% of DPSCs in conditioned medium from the damaged retina expressed the photoreceptor marker rhodopsin. These findings point to a novel and intriguing mechanism with potential therapeutic implications, highlighting the necessity of additional research in this field. Mead et al. found that DPSCs secreted a large number of NTF, which enhanced neural βIII-tubulin+ retinal cell proliferation and lengthened the neuritis.^[59] In addition, transplantation of DPSCs into the vitreous humor of mice after optic nerve injury promoted Brn-3a+ retinal ganglion cell survival and axonal regeneration. It has been reported that 44% of DPSCs expressed a photoreceptor marker rhodopsin in a conditioned medium from the damaged retina. This promising novel mechanism should be further explored for clinical applications.^[53]

2. Therapies using translocator protein (TSPO) ligands have been found to be very useful in many central and peripheral injury models.^[60] In a recent study, neuroinflammation in a mouse model of prion-induced chronic neurodegeneration (ME7) was controlled by a colony-stimulating factor receptor 1 inhibitor through TSPO modulation.^[61]

3. Other cell culture experiments also targeted Parkinson’s dementia, including the development of a unique procedure for effectively differentiating hiPSCs into high-purity midbrain dopaminergic (mDA) neurons. This breakthrough holds potential for studying the causes of neurological illnesses, conducting drug screening investigations, and using hiPSC-derived mDA neurons for cell transplantation.^[62]

4. Another study concentrated on the metallothionein (MT) family, which includes MT1 through MT4. MT1 and MT2 are present largely in astrocytes and are thought to aid in neuronal survival and axonal regeneration. Exogenous MT1 and MT2 experiments revealed enhanced neuronal survival and axonal development in cortical, hippocampal, and dopaminergic cultures.^[63]

5. Neuroprotection through G protein-coupled receptor (GPR)37/GPR37L1: Prosaptide, which acts on the receptors GPR37 and GPR37L1, has been shown in studies to protect cultured astrocytes from oxidative stress, indicating a potential function in neuroprotection. Ongoing research aims to understand how these receptors regulate astrocytic function and, as a result, neuronal activity and survival. It is yet unclear if these effects are limited to specific neuron subtypes, such as catecholaminergic neurons. Notably, small molecule GPR37 and GPR37L1 ligands are currently unavailable, leaving the pharmacology of these receptors as an unknown territory with the potential to provide significant therapeutic medicines.^[63]

6. In mice, bone marrow-derived mesenchymal stem cells (BM-MSCs) suppressed chronic experimental autoimmune encephalomyelitis (EAE), a model of MS. These cells provided neuroprotection and preserved the majority of axons in the central nervous system of successfully treated animals, showing their therapeutic potential in the context of EAE and, by extension, MS.^[25]

7. iPS cells have shown potential in the treatment of SCI patients by restoring motor function without tumorigenicity.^[64] However, challenges such as establishing iPS cells, inducing them into neural stem/progenitor cells (NS/PCs) in vitro, and quality control are significant. A collaborative team is planning iPS-based cell therapy for SCI patients in the subacute phase using clinical-grade integration-free human iPS cell lines. Clinical trials are planned for chronic phase SCI and stroke, with the aim of using iPS cell-derived NS/PC stocks for regenerative medicine.^[65]

8. Another study looked into the use of mesenchymal stromal cells (MSCs) to treat neuroinflammation in children with ASD. Children in an open-label phase I study received intravenous infusions of hCT-MSCs. These therapies were well-tolerated, with only a few occurrences of agitation during administration. Some youngsters produced anti-HLA antibodies, which did not cause any clinical problems. Notably, six of the 12 participants improved on ASD-specific measures.

9. Furthermore, some research has suggested that HSCs and mesenchymal stem cells (MSCs) may be beneficial in easing ASD symptoms by reducing inflammation and encouraging stem cell mobilization, proliferation, and differentiation in tissues. These findings point to hopeful improvements in the use of cell-based treatments to treat ASD, with an emphasis on safety and efficacy.^[66]

Although stem cell-based treatments have great promise for neuroregenerative therapy, there are still many obstacles to overcome before these experimental methods may be used in clinical settings. To achieve medical manufacturing requirements, one major difficulty is increasing the production of clinical-grade stem cells and differentiation methods.^[67] Our incomplete knowledge of the best cell dosages, delivery strategies, and mechanisms of action for patient safety and efficacy is another significant obstacle.^[68] Concerns about transplanted cells’ propensity to cause tumors and immunological rejection still exist.^[69]

Current research endeavors to tackle these obstacles by means of interdisciplinary partnerships that combine proficiencies in stem cell biology, biomaterials, clinical medicine, and industry. For instance, large-scale cell therapy production is being facilitated by the establishment of Current Good Manufacturing Practice (cGMP) facilities with a focus on stem cell manufacturing.^[70] In addition, to maximize cell distribution and survival after transplantation, improved biomaterial scaffolds are being developed.^[71] Mechanisms underlying stem cell variability and fate are being elucidated with the use of computational modeling techniques.^[72] To turn the promise of stem cell technology into practical regenerative treatments, advancements in these fields will be essential.

CONCLUSION

This review emphasizes how greatly advanced cell culture methods have the potential to change neuroregenerative therapy for severe neuropsychiatric illnesses. Our understanding of disease causes is accelerated by techniques such as 3D culture, organoids, coculture systems, microfluidics, and nanofiber scaffolds, which allow for enhanced modeling of human brain anatomy and function in vitro. The reviewed research demonstrates how these technologies can be used to test drug candidates, find new therapeutic targets, and create stem cell-based treatments for diseases that were previously thought to be incurable.

Although the emphasis here is on applications in neurologic conditions such as Alzheimer’s, stroke, and SCI, as our understanding of the underlying biological processes deepens, these state-of-the-art cell culture techniques will probably also spur advancements in the treatment of psychiatric disorders. Further exciting developments in the field of cell-based therapy for neurologic and mental illnesses include the use of DPSCs, iPSCs, and ex vivo modified cell transplantation.

There is a promise for solving and treating these urgent health issues thanks to the quick advancements in in vitro modeling and neuronal healing that this study highlights. Although stem cell-based therapies have enormous promise, more work has to be done to convert these experimental strategies into secure and efficient therapeutic interventions. Upgrading stem cell production, streamlining delivery systems, and clarifying mechanisms of action continue to be major obstacles. These obstacles will be addressed, and the potential of stem cell technologies for neuroregenerative medicine will be realized with the aid of devDevelopments in manufacturing, biomaterials, and computational modeling. Also, to fully achieve the potential of neuroregenerative medicine, more research is needed at the nexus of stem cell biology, tissue engineering, and neuropsychiatry.