Types Of Culture Conditions Research Articles

Advances in Engineered Three-Dimensional (3D) Body Articulation Unit Models

Indeed, the body articulation units, commonly referred to as body joints, play significant roles in the musculoskeletal system, enabling body flexibility. Nevertheless, these articulation units suffer from several pathological conditions, such as osteoarthritis (OA), rheumatoid arthritis (RA), ankylosing spondylitis, gout, and psoriatic arthritis. There exist several treatment modalities based on the utilization of anti-inflammatory and analgesic drugs, which can reduce or control the pathophysiological symptoms. Despite the success, these treatment modalities suffer from major shortcomings of enormous cost and poor recovery, limiting their applicability and requiring promising strategies. To address these limitations, several engineering strategies have been emerged as promising solutions in fabricating the body articulation as unit models towards local articulation repair for tissue regeneration and high-throughput screening for drug development. In this article, we present challenges related to the selection of biomaterials (natural and synthetic sources), construction of 3D articulation models (scaffold-free, scaffold-based, and organ-on-a-chip), architectural designs (microfluidics, bioprinting, electrospinning, and biomineralization), and the type of culture conditions (growth factors and active peptides). Then, we emphasize the applicability of these articulation units for emerging biomedical applications of drug screening and tissue repair/regeneration. In conclusion, we put forward the challenges and difficulties for the further clinical application of the in vitro 3D articulation unit models in terms of the long-term high activity of the models.

Mechanically tunable photo-cross-linkable bioinks for osteogenic differentiation of MSCs in 3D bioprinted constructs

3D bioprinting technique renders a plausible solution to tissue engineering applications, mainly bone tissue regeneration, which could provide the microenvironment with desired physical, chemical, and mechanical properties. However, the mechanical and structural stability of current natural polymers is a critical issue in the fabrication of bone tissue-engineered scaffolds. To overcome these issues, we have developed 3D bioprintable semi-synthetic polymers derived from natural (sodium alginate, A) and synthetic (polyethylene glycol, PEG) biopolymers. In order to enhance the cross-linking properties and biocompatibility, we have functionalized these polymers with acrylate and methacrylate chemical moieties. These selected combination of natural and synthetic polymers improved the mechanical strength due to the synergistic effect of covalent as well as ionic bond formation in the hydrogel system, which is evident from the tested tensile data. Further, the feasibility of 3D bioprinting of acrylate and methacrylate functionalized PEG and hydrogels have been tested for the biocompatibility of the fabricated structures with human umbilical cord mesenchymal stem cells (UMSCs). Further, these bioprinted scaffolds were investigated for osteogenic differentiation of UMSCs in two types of culture conditions: namely, i) with osteoinduction media (with OIM), ii) without osteoinduction media (w/o OIM). We have examined the osteoinductivity of scaffolds with the activity of alkaline phosphatase (ALP) content, and significant changes in the ALP activity was observed with the stiffness of developed materials. The extent osteogenic differentiation was observed by alizarin red staining and reverse transcription PCR analysis. Elevated levels of ALP, RUNX2 and COL1 gene expression has been observed in without OIM samples on week 1 and week 3. Further, our study showed that the synthesized alginate methacrylate (AMA) without osteoinduction supplement with young's modulus of 0.34 MPa has a significant difference in ALP quantity and gene expression over the other reported literature. Thus, this work plays a pivotal role in the development of 3D bioprintable and photo-cross-linkable hydrogels in osteogenic differentiation of mesenchymal stem cells.

Implementation of a dynamic culture condition to the heterotypic 3D breast cancer model.

Cell culture system is used for a wide range of research and biotechnology production. Majority of in vitro cell studies are conducted as static, two dimensional (2D) dish culture system where cells grow in a monolayer. However, to better reflect the in vivo condition, three dimensional (3D) culture systems were introduced that allow investigating the cell-cell and cell-microenvironment interactions. In this work, the 3D breast cancer model was investigated. Previously, we developed a 3D breast cancer model that constituted of fibroblasts and breast cancer cells seeded on the silkworm silk scaffold. The dynamic culture condition that provides the medium flow and shear forces was implemented to the model. The dynamic conditions were compared to the static cultivation regarding its influence on the number of cells, their viability, scaffold penetration, and cells co-localization. The implication of the dynamic condition to the 3D cultures resulted in a higher number and viability of the cells compared with the static 3D cultures. In contrast to the static culture condition, during the dynamic cultivation cells penetrated entirely and evenly the inner parts of the scaffold. Moreover, in coculture, the transitions like a ratio of fibroblast to the cancer cells, fibroblast morphology, and their localization were similar in both types of culture conditions, but they proceeded much faster during the dynamic cultivation. The implementation of dynamic culture condition shortened the time needed to establish the settle 3D breast cancer model. The established dynamic cancer model can be used to study tumor biology and drug screening.

Dietary Effect of Kelp (Saccharina japonica) on Gonad Quantity and Quality in Sea Urchins (Mesocentrotus nudus) Collected from a Barren Before the Fishing Season

The sea urchin Mesocentrotus nudus is commercially harvested between June and August in northeastern Japan. A previous study showed that feeding on Saccharina japonica between April and June resulted in gonad quantity and quality improvement in this species from a barren. No information is available on the optimal culture period for harvest before the fishing season. The purpose of this study was to determine whether gonad size and quality (color, texture, and taste) can be improved before the fishing season and to verify the possibility of an early harvest. The sea urchins M. nudus were collected from a barren and placed under two types of culture conditions: suspended cages in a bay and a laboratory circulation system. They were fed S. japonica between December and May or March. Gonad indices, hardness, and color [L*, a*, and b*, and ΔEab* (a unit of acceptable commercial color difference from a fishing ground for each treatment)], and free amino acid (FAA) contents of the urchin gonads at the start of rearing experiments and at the end of cage (C) and laboratory feeding (LF) cultures were measured. These variables were also compared between LF and laboratory-starved urchins to confirm the dietary effect of this kelp. Gonad indices in treatments C and LF were significantly higher than those at the start of the experiment and those in laboratory starvation (P < 0.01), although there was no significant difference between C and LF. Gonads in treatment C were significantly softer than that at the start of the experiment (P < 0.0001). ΔEab* of gonads in treatment C was the lowest, indicating that the gonad color was close to the preferred color. There was no significant difference in the total FAA content between C and LF treatments, whereas, of the umami FAA, glutamic acid content was significantly higher in gonads from C than LF. Of the bittertasting FAA, tyrosine and valine contents were significantly higher in LF gonads than in C gonads. These results suggested that gonad size, hardness, and color of M. nudus were improved before the fishing season by initiating culture in December with S. japonica feeding.

Effect of patterned electrospun hierarchical structures on alignment and differentiation of mesenchymal stem cells: Biomimicking bone.

Considering the complex hierarchical structure of bone, biomimicking the micro and nano level features should be an integral part of scaffold fabrication for successful bone regeneration. We aim to biomimic the microstructure and nanostructure of bone and study the effect of physical cues on cell alignment, proliferation, and differentiation. To achieve this, we have divided the scaffolds into groups: electrospun SU-8 nanofibers, electrospun SU-8 nanofibers with UV treatment, and micropatterned (20μm sized ridges and grooves) SU-8 nanofibers by photolithography with UV treatment. Two types of culture conditions were applied: with and without osteoinduction medium. In vitro cell proliferation assays, protein estimation, alkaline phosphatase osteodifferentiation assay, live dead assay, and cell alignment studies were performed on these micropatterned nanofiber domains. Our findings show that patterned surface induced an early osteodifferentiation of mesenchymal stem cells even in absence of osteoinduction medium. An interesting similarity with the helicoidal plywood model of the bone was observed. The cells showed layering and rotation along the patterns with time. This resembles the in vivo anisotropic multilamellar bone tissue architecture thus, closely mimicking the subcellular features of bone. This might serve as a smart biomaterial surface for mesenchymal stem cell differentiation in therapeutics where the addition of external chemical factors is a challenge.

The Metabolomic Profile of Spent Culture Media from Day-3 Human Embryos Cultured under Low Oxygen Tension.

Despite efforts made to improve the in vitro embryo culture conditions used during assisted reproduction procedures, human embryos must adapt to different in vitro oxygen concentrations and the new metabolic milieu provided by the diverse culture media used for such protocols. It has been shown that the embryo culture environment can affect not only cellular metabolism, but also gene expression in different species of mammalian embryos. Therefore we wanted to compare the metabolic footprint left by human cleavage-stage embryos under two types of oxygen atmospheric culture conditions (6% and 20% O2). The spent culture media from 39 transferred and implanted embryos from a total of 22 patients undergoing egg donation treatment was analyzed; 23 embryos came from 13 patients in the 6% oxygen concentration group, and 16 embryos from 9 patients were used in the 20% oxygen concentration group. The multivariate statistics we used in our analysis showed that human cleavage-stage embryos grown under both types of oxygen concentration left a similar metabolic fingerprint. We failed to observe any change in the net depletion or release of relevant analytes, such as glucose and especially fatty acids, by human cleavage-stage embryos under either type of culture condition. Therefore it seems that low oxygen tension during embryo culture does not alter the global metabolism of human cleavage-stage embryos.

Osteoinduction and survival of osteoblasts and bone‐marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine.

Impact of different culture conditions on growth and biopigments of Anacystis (Synechococcus) nidulans

The present study makes a comparative investigation of different commercially important pigments in A. nidulans under different culture conditions. Temperature ranges, i e 25–29 °C and 29–36 °C were provided to the cultures under three different types of culture conditions, i e constant light, alternate light and dark condition (12:12) and natural day light condition. Cultures at temperature 29–36 °C receiving alternate light and dark period (12:12) showed maximum growth with biopigment accumulation and minimum was observed in constant light at 25–29 °C. Continuous protein synthesis in the dark at the expense of the stored carbohydrates might be the reason of high biliprotein and carotenoid synthesis which also increase its commercial and economic value. This proves alternate light and dark period (12:12) at 29–36 °C condition as a cost saving strategy for large scale production of biopigments.

Matrigel Improves Functional Properties of Primary Human Salivary Gland Cells

Currently, there is no effective treatment available to patients with irreversible loss of functional salivary acini caused by Sjogren's syndrome or after radiotherapy for head and neck cancer. A tissue-engineered artificial salivary gland would help these patients. The graft cells for this device must establish tight junctions in addition to being of fluid-secretory nature. This study analyzed a graft source from human salivary glands (huSG) cultured on Matrigel. Cells were obtained from parotid and submandibular glands, expanded in vitro, and then plated on either Matrigel-coated (2 mg/mL) or uncoated culture dish. Immunohistochemistry, transmission electron microscopy, quantitative real-time-polymerase chain reaction, Western blot, and transepithelial electrical resistance were employed. On Matrigel, huSG cells adopted an acinar phenotype by forming three-dimensional acinar-like units (within 24 h of plating) as well as a monolayer of cells. On uncoated surfaces (plastic), huSG cells only formed monolayers of ductal cells. Both types of culture conditions allowed huSG cells to express tight junction proteins (claudin-1, -2, -3, -4; occludin; JAM-A; and ZO-1) and adequate transepithelial electrical resistance. Importantly, 99% of huSG cells on Matrigel expressed α-amylase and the water channel protein Aquaporin-5, as compared to <5% of huSG cells on plastic. Transmission electron microscopy confirmed an acinar phenotype with many secretory granules. Matrigel increased the secretion of α-amylase two to five folds into the media, downregulated certain salivary genes, and regulated the translation of acinar proteins. This three-dimensional in vitro serum-free cell culture method allows the organization and differentiation of huSG cells into salivary cells with an acinar phenotype.

Effect of three different types of culture conditions on Spirulina maxima growth

Growth of Spirulina maxima was studied in three types of culture conditions with four replicates each: 1) manual aeration with natural sunlight; 2) manual aeration with artificial light; and 3) constant aeration with an aquarium compressor and artificial light. After 185 days of incubation, growth declined in the first two treatments, while in the third treatment, higher growth was observed with average optical density of 3.7 against 1.8 and 1.9 in the first and second treatment, respectively. This was probably due to the fact that under constant aeration the salts were suspended avoiding the crystallization what could cause a decrease in the availability of the necessary nutrients for the growth. Also, the constant stirring allowed all the cells to receive the same amount of light promoting photosynthesis and consequently, a larger growth and characteristic green coloration. Culture with constant aeration under artificial light should be used for S. maxima cultivation because, besides reducing labor hours, it could be a more effective method for improving the economic income.

Volatile organic compounds produced by human skin cells.

Skin produces volatile organic compounds (VOCs) released to the environment with emission patterns characteristic of climatic conditions. It could be thought that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, using gas chromatography, we answered the question of whether VOC profiles of primary cultures of human dermal fibroblasts were affected by the type of culture conditions. VOCs were determined for different types of culture, finding significant differences between skin cells grown in classical monolayer culture -2D- compared with 3D matrix immobilized cultures. This indicates that VOC profiles could provide information on the physiological state of skin cells or skin.

Volatile Organic Compounds Produced by Human Skin Cells

Skin produces volatile organic compounds (VOCs) released to the environment with emission patterns characteristic of climatic conditions. It could be thought that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, using gas chromatography, we answered the question of whether VOC profiles of primary cultures of human dermal fibroblasts were affected by the type of culture conditions. VOCs were determined for different types of culture, finding significant differences between skin cells grown in classical monolayer culture -2D- compared with 3D matrix immobilized cultures. This indicates that VOC profiles could provide information on the physiological state of skin cells or skin.

Production of medaka chimeras from in vitro cultured embryonic cells

In order to develop an experimental technology employing fish embryonic stem (ES) cells, we attempted to culture cells dissociated from blastula-stage embryos of the medaka (Oryzias latipes). Three types of culture conditions were tested with regard to the culture medium, coating of the culture dishes or addition of conditioned medium. Cells incubated for 1 day under each set of culture conditions were transplanted into recipient embryos to examine whether they retained the ability to form chimeric fish, which was judged according to the body pigmentation. In a control experiment in which intact blastomeres were transplanted, chimeras were produced at a frequency of 32% among survivors. When isolated embryonic cells were dissociated and incubated in a culture medium, most of the cells did not attach to the culture dishes. The frequency of chimera formation by these cells was 15%. Use of gelatin + poly-lysine-coated dishes improved the cell attachment and the cells formed aggregated colonies, but the frequency of chimera formation by these cells decreased to 7%. Further addition of conditioned medium enhanced the cell attachment, and caused the cells to spread individually over the dishes. However, the ability of the cells to form chimeras was lost following this treatment. The results of the present study demonstrate that it is possible to generate viable chimeras from cultured embryonic cells, although further improvement of the culture conditions is necessary to develop a method for culturing fish ES cells.

Photophysiological variability of microphytobenthic diatoms after growth in different types of culture conditions

R.M. Forster and V. Martin-Jézéquel. 2005. Photophysiological variability of microphytobenthic diatoms after growth in different types of culture conditions. Phycologia 44: 393–402.Microphytobenthic diatoms have great ecological importance in estuarine and coastal marine ecosystems, yet many aspects of their physiology have not been investigated under controlled conditions. This work describes patterns in growth rates and photosynthesis in different types of culture for several species of benthic diatom. Cells were grown as epipelic biofilms both in batch culture vessels and in a continuous culture system, and differences in growth and photosynthetic physiology were measured using optical techniques. Consistent differences were observed in the maximum photochemical efficiency of PS II, Fv/Fm between species of diatoms, with highest values recorded early in the development of a culture. The maximum biomass produced also varied greatly between species, with the densest biofilms being formed by Amphora coffeaeformis. Batch cultures of benthic diatoms showed only small changes in Fv/Fm, but large decreases in the maximum rate of photosynthetic electron transport during the first week of culture development because conditions within the biofilm became unfavourable. A flow-through culture chamber was developed in order to increase the supply of nutrients to the biofilm and prevent the build-up of oxygen. In this system, high growth rates and a high maximum rate of photosynthesis was observed for periods of up to 1 wk. The flow chamber system could therefore be used for producing suitable material for comparative photophysiological experiments.

Adenovirus Mediated Gene Transduction of Primarily Isolated Mouse Islets

Expansion of pancreatic islet cell populations, especially the beta cells, using a currently available ex vivo gene transfer technology is important to develop cell therapies to treat Type I diabetes. In this study, we evaluated adenovirus mediated gene transfer efficiency in primarily isolated mouse islet cells in two types of culture conditions: freshly isolated suspended islets and cultured islets with monolayer formation. A recombinant replication deficient adenovirus vector encoding a green fluorescence protein (GFP) cDNA, Ad/ CMV-GFP, was used in the present transduction experiments. Rat 804G derived extracellular matrix (804G-ECM) and 3-isobutyl-1-methylxanthine (IBMX) were used to facilitate monolayer formation of the isolated mouse pancreatic islets. Suspended islets were transfected with Ad/CMV-GFP at more than 95% efficiency. However, analysis of immunohistochemical stains for insulin and glucagon in thin sliced sections of the islets revealed that GFP expression was localized just in the outer cells of the islets, almost all of which were glucagon positive alpha-cells. The beta-cells existing at the inner area of the suspended islets were GFP negative. In contrast, under the condition of the islets in monolayer formation cultures, all of the islet cells including the beta-cells were efficiently infected with Ad/CMV-GFP. To achieve an efficient adenoviral gene transfer to the pancreatic beta-cells, monolayer formation of the islets is critical. Such culture conditions were facilitated by combining 804G-ECM with IBMX.

ADENOVIRUS-MEDIATED GENE TRANSDUCTION OF PRIMARILY ISOLATED MOUSE ISLETS

Purpose: Expansion of pancreatic islet cell populations, especially the beta-cells, using a currently available gene transier technology is important to develop cell therapies, such as islet transplantation and a bioartificial pancreas. Thus, we evaluated adenovirus-mediated gene transfer efficiency in primarily isolated mouse islet cells in two types of culture conditions. Methods: Mouse pancreatic islet cells were isolated with a standard collagenase digestion method. A recombinant replication deficient adenovirus vector encoding a green fluorescence protein (GFP) cDNA, Ad-GFP, was used in the present transduction experiments. The 804G extracellular matrices (804G-ECM) and isobutyl methylxanthine (IBMX) were utilized to facilitate monolayer formation of isolated islets. The islet cells in floating and monolayer cultures were transduced with Ad-GFP. GFP expression of the cells was analyzed by confocal microscopy and immunochemical stain for insulin and glucagon. Results: Only outer cells of the islets in floating cultures, which were glucagon-positive, showed GFP expression. Beta-cells existing the inner area of the islets were negative for GFP activity. In contrast, beta-cells were efficiently transduced with Ad-GFP in monolayer formation cultures. Conclusion: Monolayer formation of the islets is critical to achieve an efficient adenoviral gene transfer in beta-cells. Combination of 804G-ECM with IBMX allowed efficient monolayer formation of primarily isolated mouse islets.

Differentiation of pinopsin-immunoreactive cells in the developing quail pineal organ: an in-vivo and in-vitro immunohistochemical study.

The avian pineal organ contains several types of photoreceptors with different photopigments: rhodopsin, iodopsin, and pinopsin. We have previously examined the differentiation of both rhodopsin-like and iodopsin-like immunoreactive cells during pineal development in quail embryos to determine the onset of synthesis of specific proteins and their cellular localization. In the present study, we have performed pinopsin immunohistochemistry on in-vivo developing and in-vitro cultured pineal organs of quail embryos. The results were compared with those obtained with rhodopsin and iodopsin immunohistochemistry. In the developing pineal organs, pinopsin immunoreactivity was detected at embryonic day 8, i.e. five days earlier than rhodopsin-like and iodopsin-like immunoreactivities. It was localized exclusively in the protrusions extending into the lumen throughout development, whereas rhodopsin-like and iodopsin-like immunoreactivities were usually found both in cell bodies and processes. These differences were also observed under two different types of culture conditions (dissociated cell culture and organ culture) indicating that, in the avian pineal organ, the expression pattern of the pinopsin gene is basically different from those of the other two pineal photopigments. The present study suggests that pineal cells have a mechanism for the polarized transport of pinopsin molecules.

Autologous Tumor-Specific Cytotoxicity of Tumor-Infiltrating Lymphocytes Derived from Human Renal Cell Carcinoma

Conditions for generating and expanding cytotoxic tumor-specific, tumor-infiltrating lymphocytes (TIL) were studied to improve the efficacy of adoptive cancer immunotherapy. Thus, we have examined the growth and cytolytic patterns of bulk culture TIL from human renal cell carcinoma (RCC) cultured in low (20 U/ml) or high (1,000 U/ml) dose interleukin (IL)-2, with or without irradiated autologous tumor stimulation. By 55 days in culture, TIL grown in the presence of IL-2 without tumor stimulation lost their lytic activity, whereas those exposed to tumor stimulation maintained high levels of cytotoxicity against autologous and/or nonautologous tumor targets. Only TIL grown with low dose IL-2 and autologous tumor maintained long-term (over 4 months in culture) specific cytotoxicity against the autologous tumor, even upon cryopreservation and regrowth. These TIL were 88-97% and 80% positive for CD3 and CD8, with a persistent subset exhibiting CD4+ CD8+ double positive staining. Their specific cytotoxic activity was major histocompatibility complex Class I-restricted and inhibited by pretreating the TIL with anti-CD3 monoclonal antibody. TIL exposed to the four types of culture conditions, low or high dose IL-2, with or without irradiated autologous tumors, and exhibiting different lytic specificities, all expressed mRNA for interferon-gamma and tumor necrosis factor (TNF)-alpha, but not for IL-1-beta, IL-4, IL-6, and granulocyte-macrophage colony stimulating factor. The degree of TNF-alpha mRNA expression correlated with the degree of autologous tumor-specific cytotoxicity of these TIL. This initial report demonstrates that antigen-specific cytotoxicity against the autologous tumor does, in fact, exist within the RCC tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants

Splotch (Sp) and splotch-delayed (Spd) are allelic mutations on chromosome 1 of the mouse. Embryos homozygous for either allele have neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derived structures. The fact that Spd mouse mutants sometimes have deficiencies in NCC derivatives in the absence of an NTD led to the hypothesis that neurulation and the release of NCCs may depend on a regulatory event that is common to both processes. Therefore, it may be possible to understand the cause of NTDs in these mutants by examining the basis of aberrant NCC derivatives. Caudal neural tubes were excised from day 9 Sp and Spd embryos and placed into gelatin-coated tissue culture dishes, or 3-dimensional basement membrane matrigel, and cultured for 72 hours. A cytogenetic marker was used to genotype the embryos. In planar cultures, no morphological differences were observed between NCCs from neural tube explants of Spd mutants compared to those from heterozygous or wild-type embryos. However, there appeared to be a delay in the release of NCCs from the neural tube in both Sp and Spd mutants, which was particularly evident in Sp. After 24 hours in culture, the extent of NCC outgrowth, as well as the number of NCCs emigrating from explanted neural tubes, was significantly lower in Sp and Spd mutant cultures than in controls. No differences were observed in the mitotic indices among cells which had emigrated. By 72 hours, mutant cultures and their non-mutant counterparts were similar in terms of outgrowth, cell number, and migratory capability. After 24 hours in 3-dimensional basement membrane matrigel, cell outgrowth from Sp explants was also significantly less than controls. The pattern of NCC outgrowth in both types of culture conditions indicates a 24 hour delay in mutant cultures compared to controls. This stems from a delay in the release of NCCs from the neural tube, suggesting that the defect lies within the neuroepithelium with respect to the release of NCCs.