1. Introduction
No long-term effective treatments are currently available for brain neurological disorders such as focal and global cerebral ischemia, traumatic brain injury (TBI) and neurodegenerative disorders. Current treatments are primarily focused on limiting the damage and slowing the degeneration and, most of them are only symptomatic while function restoration is rarely achieved. This underlies the need for alternative therapies such as brain cell therapy that allows functional replacement of missing or damaged neurons by transplanting cells that may differentiate into the desired phenotype and integrate the host parenchyma, or alternatively rescue the affected neuronal population. Due to the high cell death that occurs during neurodegenerative processes, brain neurological disorders are an ideal target for cell-based therapies. Several teams tried to prevent the loss of neurons or to replace them, using neuronal precursors and lately, since stem cell discovery, embryonic stem (ES) cells, adult stem cells, tissue-derived stem cells or more recently induced pluripotent stem (iPS) cells (for review see (Abeliovich & Doege, 2009; Joyce et al., 2010; Chen & Xiao, 2011; Loewenbruck & Storch, 2011). These studies demonstrated the potential of cell therapy to repair the injured brain even if poor survival of grafted cells was reported (Brundin et al., 1985; Schierle et al., 1999; Brundin et al., 2000; Isacson et al., 2003; Olanow et al., 2003). In this book chapter, brain cell therapy studies performed with adult non-transformed cells that, by nature, allow the use of autologous tissues for transplantation and overcome the immunological, availability, as well as ethical concerns will be reported.
It is important to bear in mind that, for organs with a higher level of complexity such as the brain, cell therapy remains a challenging task. Fortunately, relatively recent medical, biological and technological advances in tissue engineering approaches allow functional tissue and organ recovery by using the appropriate combination of three fundamental “tools” known as the tissue engineering triad: cells, engineered materials and signalling molecules (essentially growth factors). In practice, this is translated into a great versatility of delivery systems, which mimic the natural repairing environment of the brain tissue, creating tuneable and customized spatio-temporal gradients of signals guiding tissue regeneration. Bioactive scaffolds are likely to reinforce the success of cell replacement therapies by providing a microenvironment that facilitates the survival, proliferation, differentiation, and connectivity of transplanted and/or endogenous cells (Pettikiriarachchi et al., 2010). However, delivery of cells with scaffolds to the damaged brain still remains challenging due to practical limitations of delivery. The second part of this book chapter will provide an overview of what solutions tissue engineering may provide for adult cell therapy of the brain. Since the brain is a functionally complex organ, cell growth and differentiation alone are not enough in order to achieve its functional recovery. The correct re-establishment of the axonal connections and neuronal circuits is also necessary. In general, scaffolds for brain therapy should meet several essential requirements like being biocompatible, biodegradable, immunologically inert and able to support neurite outgrowth. The most common materials, fabrication methods and desirable properties of biomaterials used for brain protection repair and regeneration will be presented.
Brain tissue engineering has several limitations and many unanswered questions or concerns should be addressed before reaching the clinic. Moreover, with the current workflow, it takes a long time to select an effective brain tissue engineering strategy to translate into clinical studies. Additionally, recent developments in understanding the basic biology of brain tissue formation in physiological and pathological conditions have resulted in an explosion in the numbers of tissue engineering products that could be potential candidates for treating brain disorders. Screening platforms that bridge the gap between conventional tissue culture and animal models would help to improve understanding of cell-based therapies and optimize central nervous system (CNS) tissue engineering. In this regard, the usefulness of 3D brain organotypic cultures in CNS research as well as in the drug discovery process will be discussed in this book chapter.
2. Adult cell therapy for brain neuronal damages
2.1. Adult cells for cerebral ischemia and traumatic brain injury therapy
2.1.1. Adult stem cell therapy
Mesenchymal stem cells (MSCs) are the most widely investigated adult stem cells for brain cell therapy of cerebral ischemia and TBI (Table 1). Various animal models of ischemia have been used to investigate the therapeutic effects of MSCs on the lesioned brain and in all these models, bone marrow-derived MSCs resulted in an increased survival of neurons and most of the time in an improved cognitive function of the animals (reviewed in (van Velthoven et al. 2009)). In addition, MSCs have been described to reduce the thickness of the scar walls (Li et al., 2005) and may also favour angiogenesis (Chen et al., 2003; Chopp et al., 2008) as well as synaptogenesis (Chen & Chopp, 2006). Functional improvements may be observed whatever the implantation route, intravenously or intracerebrally, even if reduction in infarct volume is not always observed (Li et al., 2000). This underlines the capacity of MSCs to migrate towards lesions, as was already observed by other groups (Chen et al., 2001) after IV injection, or after intracerebral graft in a lesioned rat brain (Sykova & Jendelova, 2007; Delcroix et al., 2009). Noteworthy,
In the context of TBI, MSCs may also improve functional benefits whatever the injection route. However, a maximum of 10 % implanted MSCs may transdifferentiate into neuronal cells
2.1.2. Adult derived pluripotent stem cell therapy
A growing interest is now observed in the scientific community for the iPS cells. These “embryonic-like” cells were primarily derived from adult fibroblasts by the expression of transcription factors (a combination of 4 factors within c-Myc, KLF4, LIN28, Nanog, Oct3/4 and Sox2) (Takahashi et al., 2007; Yu et al., 2007). Several applications are currently envisioned for these pluripotent cells, which share the same potential than ES cells, without the associated ethical problems. In addition to their applications for drug discovery and toxicity testing (Laustriat et al., 2010) these cells may be helpful for complex disease understanding. They are also attractive for cell therapy studies, due to the possibility to obtain a large amount of cells from various lineages and to perform autologous grafts. iPS have now been derived from several types of adult tissues, with variable efficiency, and always using refined protocols to avoid permanent genomic integration and the use of lentiviruses for transducing the cells (Okita et al., 2011; Narsinh, et al., 2011; Lee et al., 2009; Gonzalez et al., 2009; Zhou et al., 2009; Page et al., 2009; Yu et al., 2009). Indeed, uses of viral-based protocols may be at the origins of tumour formation and may also lead to further difficulties to differentiate the cells toward a given phenotype (Yu et al., 2009). The propensity of iPS to form teratomas may also be related to the tissue of origin of the cells (Miura et al., 2009). Despite their great interest, one has to keep in mind that further developments are still required to fully understand their reprogramming process (Nakagawa & Yamanaka, 2011) in order to provide a safe cell therapeutic product for the clinic (Pera, 2011). In the context of ischemic stroke, an interesting study describes the use of iPS-derived neurons and astrocytes directly injected in the damaged cortex of a stroke animal model (Chen et al., 2011), which resulted in a reduced ischemic size with functional improvements. Another study describes the
2.2. Adult cells for neurodegenerative disorder therapy
Huntington’s and Parkinson’s diseases (HD and PD respectively) have been widely studied in cell therapy programs due to the relatively small area affected by the diseases, particularly at early stages (see for reviews (Lindvall & Kokaia, 2009; Loewenbruck & Storch, 2011)). In opposition, Alzheimer’s disease (AD) would be more complex to treat due to the multiple sites of the brain affected in this disease. Moreover, no tissue engineering strategies have yet been described for AD so that we will only focus on HD and PD in the following (Table 2).
2.2.1. Adult cell therapy
An increasing number of studies performed with foetal tissue, ES cells or NSC grafts, mostly in quinolinic acid (QA)-induced animal models of the disease (Clelland et al., 2008) gave successful results leading to clinical trials with foetal-derived cells
One of the first cell therapy strategies to treat PD has been performed with adult cell types synthesizing dopamine or its precursor L-DOPA, in order to replenish the striatum level of dopamine (for review (Drucker-Colin & Verdugo-Diaz, 2004; Fernandez-Espejo et al., 2005). Autologous adrenal medulla tissue were grafted in the caudate nucleus of 2 young PD patients, but observed functional improvements were transitory and mainly due to trophic effects as only 1 % of the cells synthesized dopamine (Madrazo et al., 1987). Most importantly, very few cells were detected 1 or 2 years after transplantation (Hurtig et al., 1989). Other studies were performed using cultured chromaffin cell suspensions, but few surviving cells in long term studies did not validate this approach (Drucker-Colin et al., 1999). Human retinal pigment epithelium (hRPE) cells derived from the inner layer of the neural retina are isolated from human eye bank for transplantation purposes (reviewed in (Stover & Watts, 2008)). hRPE cells may be expanded, present a tyroxine hydroxylase activity (Pawelek & Korner, 1982), and produce L-DOPA, which is synthesized as a melanin precursor, so that these cells may supplement the oral administration of L-DOPA
2.2.2. Adult stem cell therapy
Adult stem cells are expected to better differentiate and integrate the host brain compared to adult non-stem cells and are therefore widely investigated for their therapeutic potential in the context of neurodegenerative disorders. Whole bone marrow cells [i.e. hematopoietic stem cells (HSCs) & MSCs] implanted into the bilateral lesioned striatum of HD rat models, reversed functional deficits such as working memory (Lescaudron et al., 2003) even if the cell population responsible for the beneficial effects remained unknown. Noteworthy, autologous grafts may not be an appropriate strategy for the treatment of HD as transplanted cells would also carry the mutant huntingtin gene responsible for the disease. More recently, transplantation of MSCs, either intracerebrally or intravenously, resulted in a decreased atrophy of lesioned rat striatum (Amin et al., 2008) and in some functional benefits (Edalatmanesh et al., 2009) even though only a fraction of cells (1 %) expressed neural phenotypes. Thus, it was suggested that MSCs, by producing growth factors, allowed surviving cells within the caudate nucleus to function more efficiently and to facilitate other compensatory responses (Dunbar et al., 2006). In this regard, another study demonstrated the importance of factors such as stem cell factor (SCF), produced
In the context of PD, several teams, including ours, reported the neuronal differentiation of human MSCs toward a dopaminergic phenotype
NSCs isolated from the adult brain have been evaluated for the treatment of PD and HD due to their potential to differentiate into neurons
2.2.3. Adult cell-derived pluripotent stem cell therapy
As for several other disorders, iPS cell have been evaluated for PD cell therapy (Table 2). Dopaminergic neurons have efficiently been derived from iPS cells, and those cells have led to functional recovery in an animal model of PD (Wernig et al., 2008; Swistowski et al., 2010). In addition, it was recently demonstrated that iPS derived from patients suffering from idiopatic PD, after dopaminergic differentiation
To conclude this section, it appears clearly that MSCs were, and still are, the most widely investigated adult cells for brain cell therapy. However, their neuronal differentiation potential remains very low or uncertain after transplantation, explaining the scientific interest of the recently discovered iPS cells. In addition, the poor cell survival and engraftment observed when using chromaffin cells, hRPE cells, MSCs and in general all kinds of transplanted cells, has called into question the efficacy of a cell therapy procedure. These issues may now be acknowledged by tissue engineering approaches, discussed in the following section.
3. Combined use of adult stem cells and scaffolds for cell delivery and regeneration of CNS disorders
It is now widely admitted that cell survival, differentiation, and more generally behaviour of cells
3.1. Scaffold requirements for brain cell therapy
Implementation of tissue engineering in combination with adult cells for brain therapy is an emerging field and many requirements need to be taken into account in order to produce an appropriate tissue engineered product. The first consideration in scaffold tailoring for brain is size, but scaffolds must also be fully biodegradable and biocompatible, minimizing macrophage and microglial reaction, without inducing neurotoxicity. Scaffolds must be small enough to be easily implanted into the skull cavity in discrete and precise areas of the brain without perturbing the brain 3D organisation as observed with 30 μm microspheres (Veziers et al., 2001). Moreover, small-sized scaffolds render repeated implantations possible by stereotaxy, with no need for open-surgery (Menei et al., 2005). In this sense, microstructured and nanostructured scaffolds, produced by various techniques may be used (reviewed in (Seidlits et al., 2008)).
Scaffolds are based either on natural or on synthetic biomaterials used alone or in mixtures, providing scaffolds with different properties (see for review (Potter et al., 2008; Dalton & Mey, 2009). Scaffolds based on biodegradable gels encapsulating various molecules and cells have been studied, with e.g. polyethylene glycol (PEG) (Namba et al., 2009), diblock copolypeptide (Yang, Song et al., 2009) or hyaluronic acid (Wang & Spector, 2009) hydrogels. However, gel-based scaffold strategies most of the time require open-surgery, unless an
The adverse host cell response, such as glial scar and inflammation, occurring after scaffold implantation have to be minimized (see for review (Fournier et al., 2003)) therefore rendering the biocompatibility and biodegradability of the biomaterials a crucial parameter (Vert, 2009; Yang, Song et al., 2009). For example, implantation of PLGA microspheres into the brain does not induce a specific astrocytic or macrophage/microglia reaction, which is similar to the one observed after control fluid injection. Moreover, PLGA microspheres fully degrade into CO2 and H2O (Menei et al., 2005). Scaffolds should be able to degrade with time, with degradation products that may also be eliminated by the host, allowing a full integration of transplanted cells into the brain. This criteria was not observed for synthetic poly(methylidene malonate 2.1.2) microspheres implanted into rat brains even if biocompatibility of the intact microspheres was satisfactory (Fournier et al., 2006). It is interesting to note that size of particles may also affect the extent of the host response.
Cell attachment to the biomaterial critically depends on its surface charges, cells being attracted to positive charged surface due to sialic acid residues on the cell membrane which produce a net negative charge on the cell surface. Furthermore, the first step following implantation of a scaffold within the brain is its coverage by a non-specific layer of proteins, which may contribute to the inflammation process and biocompatibility problems (Fournier et al., 2003). Surface characteristics (charge, hydrophilicity and hydrophobicity) are therefore important points to consider when designing a new type of scaffold. Outer but also inner topography of scaffolds may affect cell behaviour once implanted into the brain. For example, the presence of pores and channels on the surface of synthetic poly--caprolactone (PCL) scaffolds may enhance host astrocytic infiltration and affect host cell migration (Wong et al., 2008). Moreover, access to nutrients is a critical parameter for neuronal cells, which require large amounts of nutrients such as glucose. Therefore, if larger implants are used, vascularisation is required for cell survival. In this sense, porous scaffolds, or scaffolds that become porous after implantation during degradation, may alleviate vascularisation problems.
Finally, a very interesting study first described the effects of matrix elasticity to direct MSCs lineage specification. Soft type matrices mimicking brain being neurogenic while stiffer matrices appeared to be myogenic and furthermore osteogenic. The observed phenotypic specification was irreversible after several weeks in culture, therefore reflecting neuronal commitment of the MSCs cultivated on soft matrices (Engler et al., 2006). Noteworthy, new data from this team seem to indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSCs behaviour (Tse & Engler, 2011). Again, this underlines the importance of the choice of the biomaterial for brain tissue engineering.
3.2. Scaffold design and manufacture
The following section reviews materials and fabrication methods used in the development of scaffolds to enhance brain tissue regeneration.
3.2.1. Materials
One of the first considerations when designing a scaffold for brain tissue engineering is the choice of the material. Some of the aspects that should be carefully taken into account are:
if the material maintains an appropriate shape after implantation,
if sterilization of the scaffold prior to implantation is possible,
if the scaffold is brain biocompatible to avoid abnormal immune responses,
if the material has the appropriate degradation rate for the desired application and
the ability to provide a controlled release of the drug, in case of materials encapsulating a therapeutic molecule such as growth factors.
Materials used in the development of scaffolds for brain tissue engineering can be broadly divided into biodegradable and non-biodegradable. Due to the transient nature of the biodegradable polymers, they are preferred for a brain application because they do not require surgical removal when the treatment is finished. Depending on their nature they can be classified in natural and synthetic materials. Both of them used alone or in combination have been tested in clinical practice. Natural materials are very interesting because they contain sites for cell adhesion, allowing for cell infiltration. These natural materials also exhibit similar properties to the soft tissues they are replacing. However, since these materials are obtained from natural sources, they must be purified before use and it is difficult to control the homogeneity of product between batches. Synthetic materials in contrast, have a known composition and can be designed to minimize the immune response or the degradation rate. Materials (natural and synthetic) used in brain tissue engineering applications, with their benefits and drawbacks will be discussed below in this section.
3.2.1.1. Natural materials
3.2.1.2. Synthetic materials
3.2.2. Scaffold fabrication techniques
As brain tissue engineering progresses, the need of novel scaffold structures and fabrication techniques has become of great importance (See Walker et al., 2009; Subia, 2010). The most common scaffold production methods are revised below. After fabrication, all of these scaffolds are then characterized in terms of morphology, mechanical, bulk and surface properties.
3.3. Overview of strategies used to combine adult stem cells and scaffolds
The following section reviews primary studies that investigated the potential of different combinations of cells and scaffolds to improve the cell therapy benefits for PD, HD, cerebral ischemia and TBI. Studies on hRPE cells in combination with gelatine-based scaffolds, a device that underwent a phase II clinical trial for the treatment of PD will also be described. Several strategies are now focusing on improving cell interactions with the biomaterials by modifying its surface using biological molecules, mainly derived from ECM, to better regulate grafted cell behaviour. After briefly describing the molecular mechanisms of cell- ECM molecule interactions and its effect on cell behaviour, we will present some studies using such “biomimetic scaffolds”. Finally, the PAMs developed in our laboratory, that constitute a more advanced approach combining a bioactive surface with the controlled delivery of a growth factor will be presented.
3.3.1. Gel-based scaffolds and cell encapsulating technology
By contributing to maintain brain’s integrity as well as by favouring the integration of host cells inside the marginal cavities, gel-based scaffolds, without cells, may be advantageous to repair the brain after an ischemic stroke (Yamashita et al., 2009). Moreover, differentiated iPS injected together with a fibrin glue under the dura mater enhanced the effect of these iPS injected alone, which led to a reduced infarct volume as well as to functional recovery after an ischemic stroke; thereby underlining the importance of a support for the cells (Chen & Xiao, 2011).
In the context of TBI, degradable collagen scaffolds did not reduce the lesion size nor did they improve functional recovery, unless if seeded with hMSCs (Lu et al., 2007). Indeed, four days after TBI, transplantation of a cylindrical collagen scaffold seeded with hMSCs in the lesion cavity induced a reduction in the lesion volume, together with an improved spatial learning and sensorimotor function of the animals. Recently, the same team reported that delayed transplantation of these complexes (7 days after TBI) further enhanced spatial learning and sensorimotor function, and induced angiogenesis in the injured cortex as well as transcallosal fiber length (Qu et al., 2009; Xiong et al., 2009). In experimental models of TBI, MSCs are usually injected adjacent to the lesion within the parenchyma to avoid injection within the lesion cavity. An advantage of this gel-based strategy was the possible use of 3 fold more MSCs compared to parenchymal injection, therefore increasing the regenerative potential. However, the major limitation was the need for open-surgery to implant the device. This issue may potentially be addressed using an
3.3.2. Cell adhesion on particulate scaffolds
As previously described, the major problems encountered when grafting chromaffin cells in the context of PD was the poor cell survival and the absence of long term effects
A similar strategy combines cultured hRPE cells attached to biocompatible, non biodegradable cross-linked porcine gelatine microcarriers, with a mean diameter of 100 µm. Several preliminary studies in parkinsonian rat models (unilateral and bilateral lesions) proved the efficacy of these grafted complexes, named Spheramine® (Watts et al., 2003). An increased survival of hRPE cells, without immunosuppression, and long term functional improvements were observed, although chronic inflammation was reported at later time-points (5 months) (Flores et al., 2007). These microcarriers have also been implanted in the brain of hemi-parkinsonian monkeys and resulted in long term cell survival and functional improvements at 18 months (Doudet et al., 2004). As expected, hRPE cells unattached to microcarriers did not survive well in the brain, and did not produce a lasting therapeutic effect in various PD animal models. These encouraging results led to an open-label clinical study that included 6 patients with advanced PD receiving 325,000 hRPE cells attached to microcarriers and demonstrated a good tolerability to Spheramine®. Moreover, at 6 months post-operation, the mean UPDRS-M (off) score improved to 34 % from the pre-operation baseline. Half of the patients also demonstrated a reduced Dyskinesia Rating Scale scores (Bakay et al., 2004). The success of this strategy finally led to a phase II double-blind, randomized, multicenter, placebo-controlled (sham surgery) study to evaluate safety, tolerability, and efficacy of Spheramine® implanted bilaterally into the postcommissural putamen of patients with advanced PD, even if the mechanisms underlying the improved survival of hRPE cells upon attachment to gelatin are not yet well elucidated (Stover & Watts, 2008). Unfortunately, preliminary results of the phase II clinical trial seem to indicate that the study failed to demonstrate the efficacy of Spheramine® beyond a remarkable placebo effect. A reason for that may be the lack of long term survival of the cells in the human brain, as only around 0.036 % of cells survival was observed in the brain of a patient who died 6 months after surgery (Farag et al., 2009).
Adult rat choroid plexus cells have the potential to secrete a wide range of growth factors, and have been used as candidate cells for HD cell therapy (Borlongan et al., 2008). In this study, choroid plexus cells were encapsulated in alginate beads to improve cell viability and to prevent host rejection. Transplantation of these cells into rat striatum resulted in an encouraging neuroprotection when a QA lesion was performed 3 days after cell transplantation, even if no evidence was provided concerning the specific sparing of GABAergic medium spiny projection neurons, especially sensitive to degeneration in the context of HD.
3.3.3. Molecular mechanisms of cell adhesion to scaffolds
The ECM contains adherent glycoproteins, glycosaminoglycans and ions. ECM proteins, such as collagen, FN, LM, tenascin and proteoglycans, interact with each other forming a supportive scaffold for the cells within the tissue (see for review (Bosman & Stamenkovic, 2003)). Composition and proportion of its constituents vary depending on the type of tissue, thereby giving different mechanical, chemical or signalling cues to the surrounding cells. We will here focus on FN and LM because of their astonishing variety of effects on cells as well as because of their wide use for brain tissue engineering applications. FN and LM structures have been described a few decades ago and are constituted of an assembly of large polypeptides, all of them having specific interaction sites with other ECM molecules or cell surfaces (Hynes & Yamada, 1982; Engvall & Wewer, 1996; Powell & Kleinman, 1997). LM and FN interact with cells
Potential roles of ECM molecules for brain repair are now widely admitted. Endogenous levels of FN and LM increase in a TBI context
ECM molecules may also affect proliferation as well as life span of cells, this being the rationale for expanding MSCs on ECM molecules in several
Neural precursor differentiation, by enhancing neurite outgrowth
3.3.4. The pharmacologically active microcarriers (PAMs): a tool to combine the biomimetic approach and the controlled release of a growth factor
In addition to the biomimetic approach, another way to improve the efficiency of cell grafts is to deliver a growth factor by the transplanted scaffolds, further affecting the fate of both transplanted and host cells. Interestingly, the potential of microsphere-hydrogel scaffolds to deliver 2 growth factors at specific rates has been described (Burdick et al., 2006), but has not yet led to
The proof of concept of this unique and simple device of cell and protein-delivery in neuroprotection and tissue repair for the treatment of neurological disorders has first been validated in a PD rat model using a neuronal cell line (PC12 cells) transported by nerve growth factor (NGF)-releasing PAMs. (Tatard et al., 2004). The efficacy of PAMs for cell therapy of PD in a clinical paradigm was then demonstrated using GDNF-releasing PAMs, conveying a small number of embryonic ventral mesencephalon dopaminergic cells (Tatard et al., 2007). We next implemented the PAMs transporting MIAMI cells for PD adult stem cell therapy. MIAMI cells, may differentiate toward neuronal-like cells in a neurotrophin 3 (NT3) dependent manner (Tatard et al., 2007), especially when pre-treated with EGF and bFGF (Delcroix, Curtis et al., 2010). Moreover, a LM surface enhances the neuronal differentiation (Delcroix et al., 2011). We designed PAMs made of 60 µm PLGA microspheres encapsulating NT3 and covered with a LM biomimetic surface. After adhesion of dopaminergic-induced (DI)-MIAMI cells, the PAM/cell complexes were grafted in the partially dopaminergic-deafferented striatum of rats and led to a strong reduction of the amphetamine-induced rotational behaviour together with the protection/repair of the nigrostriatal pathway. These effects were correlated with the increased survival of DI-MIAMI cells which differentiated towards dopaminergic-like cells and may also secrete growth factors (Delcroix et al., 2011). We thus showed that combining growth factors, cell adhesion molecules and an adapted 3D structure in the same polymeric scaffold allows the synthesis of an adaptable and very efficient system that can deliver stem cells and give them appropriate cues allowing better stem cell survival, differentiation and integration into the host tissues after implantation. After deeper characterization of the underlying mechanisms, this tissue engineering strategy may ultimately set the ground for pre-clinical studies with non-human primates to increase the efficiency of MSC therapy of the brain.
4. Unanswered questions in tissue engineering for targeting CNS disorders using conventional tissue culture and animal models
Although brain tissue engineering has shown a certain level of therapeutic benefits, there are still many unanswered questions and concerns that need to be addressed. For instance, one general problem found in cell therapy is the difficulty to reproduce results between laboratories. Differences on the source, method of preparation, differentiation status and age of the stem cells used may be the cause. Moreover, with the current workflow it takes a long time to select an effective stem cell strategy to translate into clinical studies. The main steps to follow include the selection of the source of cells (MSC, NSC, ESC, iPS), the choice of the culture media, the state of stem cell cycle (undifferentiated
In general, it is difficult to understand stem cell mechanisms of action and fate using conventional tissue culture or animal models due to the limitations of both systems.
5. Organotypic 3D culture models: novel platforms for optimization of CNS cell therapy and tissue engineering
Since their introduction, organotypic cultures of rat brain slices have become a useful tool to study drug effects. Brain organotypic slices are
5.1. Preparation of organotypic cultures of CNS tissue
Organotypic brain slice culture was developed by inspiration on explant cultures techniques of various anatomical origins. Since then, several methods have been developed to prepare and maintain slices alive in long-term culture. The roller-tube technique based on the use of roller tubes was first described in 1981 (Gahwiler, 1981). However, most studies now use the membrane interface method described in 1991 (Stoppini et al., 1991) which provides an easier access to the slice culture. The principle of the membrane interface method is to maintain brain slices on a porous membrane filter at the interface between medium and a humidified atmosphere. The medium provides adequate nutrition to tissues through the membrane
Briefly, the brain is dissected free of the skull and placed into a saline solution whose composition resembles that of the cerebro-spinal fluid and is thus often referred to as artificial cerebro-spinal fluid (ACSF). Slicing is performed with a vibrating microtome at high amplitude and very slow speed. Slice thickness varies according to specimen and the type of experiment, from 150 to 400 μm (Lossi et al., 2009). Over the years, slice culture systems have been successfully established from a variety of brain regions including hippocampus (far more frequently), striatum, cortex, spinal cord and cerebellum. Furthermore, a number of tissue slice co-cultures have been developed, which allow the assessment of inter-neural responses across brain regions. Moreover, when organotypic slices are exposed to certain toxic conditions (oxygen-glucose deprivation, neurotoxins, glutamate-mediated excitotoxicity) they develop many pathophysiological features found in brain disorders and consequently, brain organotypic slices can be used as ex-vivo models of CNS diseases. Nowadays, organotypic models for global cerebral ischemia, ischemic stroke, Alzheimer’s disease, PD, HD, TBI, epilepisa and amyotrophic lateral sclerosis have been described (For review see (Noraberg et al., 2005; Sundstrom et al., 2005; Cho et al., 2007; Cimarosti & Henley, 2008; Lossi et al., 2009)). Among them, hippocampal organotypic slices exposed to oxygen-glucose deprivation, a model for global cerebral ischemia is the most commonly used.
5.2. Applications
Organotypic slices have been commonly used as models to investigate mechanism and treatment strategies for neurodegenerative disorders. Interestingly, the utility of brain slices to test CNS cell therapy efficacy has recently started to be investigated. Organotypic cultures are a very useful tool for screening of candidate stem cells for a specific pathology. They also could be used to track survival, differentiation, proliferation and migration of the transplanted cells with or without microscaffolds or to study graft and host interactions (Figure 3). Stem cell mechanisms of neuroprotection or paracrine secretions of MSCs could be also studied using brain slices. Charriere et al.,(Charriere et al. 2010) analyzed the interactions between bone marrow stromal cells and hippocampal slice cultures to clarify putative cross-interactions between MSCs and the CNS that could explain the molecular mechanism of stem cells. The dopaminergic differentiation of ES in a PD organotypic model and the effect of morphogenetic proteins such as LM on the differentiation of the cells has also been studied (Kearns et al., 2006). It was also shown that olfactory ensheathing cells (OEC) when co-cultured with the auditory brain stem slice culture not only promoted neurite outgrowth from the cochlear nucleus region of the brain stem slice but also supported the OEC indicating positive interactions between both (Jiao et al. 2010). Recently, our group examined the potential of MIAMI cells injected into the hippocampus to prevent neuronal damage induced by global ischemia using rat hippocampal slices exposed to oxygen-glucose deprivation. We showed that MIAMI cells prevented neuronal damage. MIAMI cell therapeutic value was significantly increased when delivering the cells complexed with FN-coated biomimetic microcarriers probably by increasing stem cell survival and paracrine secretion of pro-survival and/or anti-inflammatory molecules as concluded from survival, differentiation and gene expression analysis (Garbayo et al., 2011). Furthermore, brain organotypic slices can be used to perform tissue biocompatiblity studies of scaffolds prepared for brain tissue engineering application and to determine how changes in the composition or in the functionalization of the scaffold could compromise their brain biocompatibility. In this context, Kristensen
An innovative approach using modified organotypic slices derived from a transgenic, mutant, null, gain-of-function, loss-of function, knock-in, or knockout animals was recently proposed (Li & Loudon, 2008). They discuss the possibility of using modified organotypic slice cultures to understand how implanted cells interact with resident cellular matrix and injured residential cells to predict their
5.3. Limitations of organotypics
Organotypic slices are not without limitations. The most limiting features are:
currently brain slices are only produced from juvenile donor animals as it is known that young tissue has more neuronal plasticity and are more resistant than older tissue. Recent reports have used older donors to prepare brain organotypic slices but although it is possible, a low yield was observed and slices remained viable for only 3 to 4 weeks.
Automation is a challenge since a skilled operator is required for the production of the slices.
Not all brain areas are amenable to culture being the most appropriate the regions with a lamellar structure that can be aligned parallel to the plan of slicing.
Organotypics do not have a functional vascular compartment (for review see Sundstrom et al., 2005).
6. Conclusions
Although the ideal brain scaffold that satisfies all the requirements does not exist yet, the past several years have seen considerable progress in this field of study. For instance, materials of many types have been used to create brain scaffolds capable of providing sustained delivery of signalling molecules and an adequate 3D support for transplanted cells, thereby increasing cell survival and even guiding cell differentiation and fate
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