Passaging protocols for mammalian neural stem cells in suspension bioreactors

Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders. As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large-scale expansion of mammalian NSC. Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media. The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time. We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter. When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 x 10(3) was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 microm) below levels at which necrosis would occur. Moreover, cell densities of 1.0 x 10(6) cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80%. The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates. We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.     

Physical passaging of embryoid bodies generated from human pluripotent stem cells

Spherical three-dimensional cell aggregates called embryoid bodies (EBs), have been widely used in in vitro differentiation protocols for human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Recent studies highlight the new devices and techniques for hEB formation and expansion, but are not involved in the passaging or subculture process. Here, we provide evidence that a simple periodic passaging markedly improved hEB culture condition and thus allowed the size-controlled, mass production of human embryoid bodies (hEBs) derived from both hESCs and hiPSCs. hEBs maintained in prolonged suspension culture without passaging (>2 weeks) showed a progressive decrease in the cell growth and proliferation and increase in the apoptosis compared to 7-day-old hEBs. However, when serially passaged in suspension, hEB cell populations were significantly increased in number while maintaining the normal rates of cell proliferation and apoptosis and the differentiation potential. Uniform-sized hEBs produced by manual passaging using a 1∶4 split ratio have been successfully maintained for over 20 continuous passages. The passaging culture method of hEBs, which is simple, readily expandable, and reproducible, could be a powerful tool for improving a robust and scalable in vitro differentiation system of human pluripotent stem cells.     

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.     

Inoculation and growth conditions for high-cell-density expansion of mammalian neural stem cells in suspension bioreactors

Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntington's and Parkinson's disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.     

Suspended in culture--human pluripotent cells for scalable technologies

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively termed human pluripotent stem cells (hPSCs), are typically derived and maintained in adherent and semi-defined culture conditions. Recently a number of groups, including Chen et al., 2012, have demonstrated that hESCs can now be expanded efficiently and maintain pluripotency over long-term passaging as aggregates in a serum-free defined suspension culture system, permitting the preparation of scalable cGMP derived hPSC cultures for cell banking, high throughput research programs and clinical applications. In this short commentary we describe the utility and potential future uses of suspension culture systems for hPSCs.     

Passaging human neural stem cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology. This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research. The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003). We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days. Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).     

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging.METHODS: Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers.RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively.CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.     

Cell cycle kinetics of expanding populations of neural stem and progenitor cells in vitro

Neural stem cells (NSCs) are undifferentiated, primitive cells with important potential applications including the replacement of neural tissue lost due to neurodegenerative diseases, including Parkinson's disease, as well as brain and spinal cord injuries, including stroke. We have developed methods to rapidly expand populations of mammalian stem and progenitor cells in neurosphere cultures. In the present study, flow cytometry was used in order to understand cell cycle activation and proliferation of neural stem and progenitor cells in suspension bioreactors. First, a protocol was developed to analyze the cell cycle kinetics of NSCs. As expected, neurosphere cells were found to cycle slowly, with a very small proportion of the cell population undergoing mitosis at any time. Large fractions (65-70%) of the cells were detected in G1, even in rapidly proliferating cultures, and significant fractions (20%) of the cells were in G0. Second, it was observed that different culturing methods influence both the proportion of neurosphere cells in each phase of the cell cycle and the fraction of actively proliferating cells. The results show that suspension culture does not significantly alter the cell cycle progression of neurosphere cells, while long-term culture (>60 days) results in significant changes in cell cycle kinetics. This suggests that when developing a process to produce neural stem cells for clinical applications, it is imperative to track the cell cycle kinetics, and that a short-term suspension bioreactor process can be used to successfully expand neurosphere cells.     

Scalable GMP compliant suspension culture system for human ES cells

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.     

A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.     

Direct Generation of Neurosphere-Like Cells from Human Dermal Fibroblasts

Neural stem cell (NSC) transplantation replaces damaged brain cells and provides disease-modifying effects in many neurological disorders. However, there has been no efficient way to obtain autologous NSCs in patients. Given that ectopic factors can reprogram somatic cells to be pluripotent, we attempted to generate human NSC-like cells by reprograming human fibroblasts. Fibroblasts were transfected with NSC line-derived cellular extracts and grown in neurosphere culture conditions. The cells were then analyzed for NSC characteristics, including neurosphere formation, gene expression patterns, and ability to differentiate. The obtained induced neurosphere-like cells (iNS), which formed daughter neurospheres after serial passaging, expressed neural stem cell markers, and had demethylated SOX2 regulatory regions, all characteristics of human NSCs. The iNS had gene expression patterns that were a combination of the patterns of NSCs and fibroblasts, but they could be differentiated to express neuroglial markers and neuronal sodium channels. These results show for the first time that iNS can be directly generated from human fibroblasts. Further studies on their application in neurological diseases are warranted.     

Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations

The concept of stem-cell plasticity received strong support from a recent observation that extensively passaged, clonally derived neural stem cells could contribute to hematopoiesis. We investigated whether hematopoietic potential was a consistent or unusual feature of neural stem cells, and whether it depended on the extent of in vitro passaging before transplantation. Here we transplanted over 128 x 10(6) neurosphere cells into 128 host animals; however, we never observed contribution to hematopoiesis, irrespective of the number of passages and despite the use of an assay that could detect the contribution of a single blood stem cell to hematopoietic repopulation. Although extensively cultured neurosphere cells continued to generate neural progeny, marked changes in their growth properties occurred, including changes in growth-factor dependence, cell-cycle kinetics, cell adhesion and gene expression. Our results exclude hematopoietic competence as a consistent property of intravenously infused neural stem cells. However, the consistent changes that occurred during extended passaging are compatible with genetic or epigenetic alterations and suggest that rare transformation events may account for the neural-to-blood fate switch originally reported.     

Neural stem cell lineages are regionally specified, but not committed, within distinct compartments of the developing brain.

Regional patterning in the developing mammalian brain is partially regulated by restricted gene expression patterns within the germinal zone, which is composed of stem cells and their progenitor cell progeny. Whether or not neural stem cells, which are considered at the top of the neural lineage hierarchy, are regionally specified remains unknown. Here we show that the cardinal properties of neural stem cells (self-renewal and multipotentiality) are conserved among embryonic cortex, ganglionic eminence and midbrain/hindbrain, but that these different stem cells express separate molecular markers of regional identity in vitro, even after passaging. Neural stem cell progeny derived from ganglionic eminence but not from other regions are specified to respond to local environmental cues to migrate ventrolaterally, when initially deposited on the germinal layer of ganglionic eminence in organotypic slice cultures. Cues exclusively from the ventral forebrain in a 5 day co-culture paradigm could induce both early onset and late onset marker gene expression of regional identity in neural stem cell colonies derived from both the dorsal and ventral forebrain as well as from the midbrain/hindbrain. Thus, neural stem cells and their progeny are regionally specified in the developing brain, but this regional identity can be altered by local inductive cues.     

A review of bioreactor protocols for human neural precursor cell expansion in preparation for clinical trials

Tissue-specific human neural precursor cells (hNPCs) can be isolated from various regions of the developing or adult central nervous system and may serve as a viable source of cells in cell replacement therapies for the treatment of neurodegenerative disorders. However, in order for cell replacement strategies to become a routine therapeutic option for the treatment of neurodegenerative disorders, hNPCs should be generated under standardized and controlled conditions. Studies over the last two decades have focused on developing cell growth media and cell handling protocols for expansion and differentiation of hNPCs in culture. Key studies have reported the development of serum-free growth media and large-scale computer-controlled suspension bioreactors that can support high cell proliferation rates (doubling times < 3 days), multipotentiality, and potential neurogenic differentiation (more than 60% neurons). Moreover, bioengineering studies have focused on controlling culture conditions in suspension bioreactors including inoculation, hydrodynamics of culture, oxygen and nutrients transfer to the cells, monitoring in situ physiological parameters using process control techniques, and expansion for extended periods of time. In addition, in vitro and in vivo characterization of hNPCs have been performed, providing information on stem/progenitor cell characteristics, cell surface analysis, and appropriate type of cells to use in transplantation studies.     

Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts were used, a mechanical method of passaging led to better cell growth than passaging by trypsin digestion. We also found that exogenous supplementation with leukemia inhibitory factor maintained the embryonic stem cell-like cells in an undifferentiated state, whereas addition of stem cell factor resulted in their differentiation. Our findings provide an experimental basis for the establishment of an effective culture system for bovine embryonic stem cells.     

Simple methods for generating neural,bone and endodermal cell types from chick embryonic stem cells

Most work on embryonic stem cell differentiation uses mammalian cells derived from the blastocyst stage and some of the most widely used protocols to induce differentiation involve growing these cells in monolayer culture. Equivalent stem cells can be obtained from embryos of non-mammalian vertebrates, but to date this has only been successful in birds. These cells can contribute to all somatic lineages in chimaeras and can be induced to differentiate into a variety of cell types in vitro via embryoid body formation. However to date there are no reliable methods for differentiating them into descendants from each of the germ layers in monolayer culture, comparable to the protocols used in mammals. Here we describe three simple and reproducible protocols for differentiation of chick embryonic stem cells into mesoderm (bone), endoderm and neuroectoderm (neurons and glia) in monolayer culture. These methods open the way for more direct comparisons of the properties of mammalian and avian embryonic stem cells that may highlight similarities and differences.     

Embryonic stem cells remain highly pluripotent following long term expansion as aggregates in suspension bioreactors

Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their potential use as the primary material in various tissue engineering applications. Successful clinical implementation of this technology would require a quality controlled reproducible culture system for the expansion of the cells to be used in the generation of functional tissues. Recently, we showed that suspension bioreactors could be used in the regulated large-scale expansion of highly pluripotent murine ESCs. The current study illustrates that these bioreactor protocols can be adapted for long term culture and that murine ESC cultures remain highly undifferentiated, when serially passaged in suspension bioreactors for extended periods. Flow cytometry analysis and gene expression profiles of several pluripotency markers, in addition to colony and embryoid body (EB) formation tests were conducted at the start and end of the experiment and all showed that the ESC cultures remained highly undifferentiated over extended culture time in suspension. In vivo teratoma formation and in vitro differentiation into neural, cardiomyocyte, osteoblast and chondrocyte lineages, performed at the end of the long term culture, further supported the presence of functional and undifferentiated ESCs in the expanded population. Overall, this system enables the controlled expansion of highly pluripotent murine ESC populations.     

大鼠脑神经干细胞系(RNSC-FMU 1)的建立和鉴定

采用无血清培养液分离和培养新生SD 大鼠脑的神经干细胞,以机械分散的方法传代,成功地建立了大鼠脑神经干细胞系(RSNC-FMU 1)。该细胞系可在体外长期传代,至今已在体外连续生长超过21个月(>100代),保持了神经干细胞的特性和正常的核型,经诱导可分化成为神经元、星形胶质细胞和少突胶质细胞,具有较旺盛的自新能力,倍增时间约为20h,并可冷冻保存,裸鼠体内移植证实其不具有成瘤性。该细胞系为神经干细胞研究提供了一个良好的工具。     

神经干细胞的分离、培养及应用前景

胚胎和成年哺乳动物脑内均存在神经干细胞,具有潜在的增值和分化能力。在一定条件下,神经干细胞可向多个方向分化,生成神经元和神经胶质细胞,这为利用神经干细胞进行中枢神经系统退行性病变和损伤的治疗打下基础。     

Expansion of human embryonic stem cells: a comparative study

Objectives: Human embryonic stem cells (hESC) are promising for tissue engineering (TE) purposes due to their unique properties. However, current standard mechanical passaging techniques limit rates of possible TE experiments, as it is difficult to obtain high enough numbers of the cells for experimentation. In this study, several dissociative solutions and application methods are tested for their applicability to, and influence on, hESC culture and expansion. Materials and methods: Expansion of two hESC lines, H1 and VUB01, subjected to different passaging techniques, was evaluated. Four dissociative solutions – TrypLE? Express, Trypsin‐EDTA, Cell Dissociation Solution and Accutase?– were combined with two application protocols. As reference conditions, manual and bead‐based passaging techniques were used. Results: Results showed that use of Cell Dissociation Solution in combination with a slow adaptation protocol, generated the best expansion profile for both cell lines. The hESC single cell lines remained pluripotent, had good expansion profiles and were capable of differentiation into representatives of all three germ layers. Reproducibility of the results was confirmed by adaptation for three other hESC lines. Conclusion: Use of Cell Dissociation Solution, combined with slow adaptation protocol, allows a fast switch from the mechanical passaging technique to a single‐cell split technique, generating stable and robust hESC cell lines, which allow for large scale expansion of hESC for TE purposes.