differentiating human neuronal stem cells into 3d culture using a porous scaffold

Differentiating Human Neuronal Stem Cells into 3D Culture Using a Porous Scaffold

Rehydrate the scaffolds by adding to each well of the Alvetex 12-well plate, 2 milliliters of 70% ethanol, prepared using water for irrigation. Avoid touching the scaffolds by dispensing the 70% ethanol down the wall of each well. Carefully aspirate the 70% ethanol and immediately wash the scaffolds with 2 milliliters per well of DMEM F12 for one minute.
Repeat the wash procedure twice. Carefully aspirate the DMEM F12 after the final wash. Coat the scaffolds by adding 2 milliliters of laminin in DMEM F12 to each well. Incubate the plate at 37 degrees Celsius in a 5% carbon dioxide humidified incubator for a minimum of two hours.
Next, wash the plate with warm DMEM F12. Seed each scaffold with approximately 500,000 human neural stem cells or HNSCs in a volume of 150 microliters of RMM medium with growth factors. Incubate the plate for three hours to allow the cells to settle into the scaffolds.
After three hours, add 3.5 milliliters of RMM to each well, taking care not to dislodge cells from the scaffolds. Incubate the plate for seven or 21 days. Replace the medium after one day, which is the first feeding, and replace the medium again 2 to 3 days after the first feeding.

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1. HNSC differentiation on Three-Dimensional (3D) Culture
NOTE: Isolate and derive human neural stem cells (hNSCs) from an ethically approved tissue described in a previous publication. Please follow the ethical and institutional guidelines while following this procedure.
<ol>
	<li>3D Culture Using&#160;12-well Plate
	<ol>
		<li>Use an aseptic technique throughout the procedure. In a biological safety cabinet, re-hydrate scaffolds by adding 2 ml/well of 70% ethanol prepared using water for irrigation (WFIr). Avoid touching the scaffolds by dispensing the 70% ethanol down the wall of each well.</li>
		<li>Carefully aspirate the 70% ethanol and immediately wash the scaffolds with 2 ml/well Dulbecco&#39;s Modified Eagle Medium F12 (DMEM: F12) for 1 min. Repeat the wash procedure twice. Carefully aspirate the DMEM: F12 after the final wash.</li>
		<li>Coat scaffolds by adding 2 ml/well of laminin (10 &#181;g/ml) in DMEM: F12. Incubate plate at 37 &#176;C in a 5% CO2&#160;humidified incubator for a minimum of 2 hr. Wash the plate with warm DMEM: F12.</li>
		<li>Seed each scaffold with approximately 500,000 hNSCs in a volume of 150 &#181;l of RMM+growth factors (GFs).</li>
		<li>Incubate the plate for 3 hr at 37 &#176;C in a 5% CO2&#160;humidified incubator to allow cells to settle into the scaffolds.</li>
		<li>Add 3.5 ml of RMM to each well, taking care not to dislodge cells from scaffolds.</li>
		<li>Incubate the plate for 7 days; replace RMM medium (3.5 ml/well) after 1 day (first feeding) and again after 2-3 days from the first feeding.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM:F12</td>
			<td>Invitrogen</td>
			<td>21331-020</td>
			<td>For RMM medium preparation</td>
		</tr>
		<tr>
			<td>Human Albumin Solution&#160;</td>
			<td>Baxter health care limited&#160;</td>
			<td>1501052</td>
			<td>For RMM medium used at a final concentration of&#160; 0.03%</td>
		</tr>
		<tr>
			<td>Transferrin, Human recombinant</td>
			<td>Sigma</td>
			<td>T8158</td>
			<td>For RMM medium used at a final concentration of&#160; 5 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Putrescine DiHCl&#160;</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td>For RMM medium used at a final concentration of&#160; 16.2 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Insulin, Human recombinant&#160;&#160;</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>For RMM medium used at a final concentration of&#160; 5 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Progesterone&#160;</td>
			<td>Sigma</td>
			<td>P6149</td>
			<td>For RMM medium used at a final concentration of&#160; 60 ng/ml</td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Invitrogen</td>
			<td>25030-24</td>
			<td>For RMM medium used at a final concentration of&#160; 2 mM</td>
		</tr>
		<tr>
			<td>Sodium Selenite&#160;&#160;</td>
			<td>Sigma</td>
			<td>S9133</td>
			<td>For RMM medium used at a final concentration of 40 ng/ml</td>
		</tr>
		<tr>
			<td>bFGF&#160;&#160;&#160;&#160;&#160;</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>To be added to RMM medium, used at a final concentration of 10 ng/ml</td>
		</tr>
		<tr>
			<td>EGF&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Peprotech</td>
			<td>AF-100-188</td>
			<td>To be added to RMM medium, used at a final concentration of 20 ng/ml</td>
		</tr>
		<tr>
			<td>4-OHT</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td>To be added to RMM medium, used at a final concentration of 100 nM</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>AMS Biotech</td>
			<td>3400-010-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for irrigation</td>
			<td>Baxter Healthcare</td>
			<td>UKF7114</td>
			<td></td>
		</tr>
		<tr>
			<td>Alvetex 12 well plate&#160;</td>
			<td>Reinnervate Ltd</td>
			<td>AVP002</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Merck&#160;</td>
			<td>1.00986.1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Stevanato, L. et al., <a href="https://app.jove.com/v/52410">Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes.</a> J. Vis. Exp. (2015)
This video showcases the development and differentiation of human neural stem cells within a three-dimensional scaffold. A laminin coating on the scaffold promotes cell attachment, while a growth factor-free neural medium induces cell differentiation into neural progenitor cells, which extend their processes within a porous architecture in multiple directions, forming a three-dimensional culture

1. HNSC differentiation on Three-Dimensional (3D) Culture
NOTE: Isolate and derive human neural stem cells (hNSCs) from an ethically approved tissue ...

Begin with a multi-well plate containing a porous insert that holds a polystyrene scaffold possessing a three-dimensional porous structure.
Add ethanol to rehydrate the scaffold and wash it with the medium.
Introduce a medium containing an extracellular matrix protein, laminin. Incubate to allow laminin to coat the scaffold.
Seed the human neural stem cells in a neural medium containing growth factors onto the laminin-coated scaffold.
Incubate to allow the stem cell migration into the porous scaffold where the laminin promotes cell adherence to the scaffold.
The cells utilize the growth factors and proliferate within this scaffold.
Add a growth factor-free neural medium and regularly replace the spent medium to maintain cell viability.
The absence of growth factors stimulates the differentiation of stem cells into neural progenitor cells.
Over time, the neural processes of these progenitor cells elongate, allowing multidirectional cell growth and forming a three-dimensional culture.

13 次观看. 使用多孔支架将人神经元干细胞分化为 3D 培养物

视频: 使用多孔支架将人神经元干细胞分化为 3D 培养物 - 实验

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

在SH-SY5Y人神经母细胞瘤分化

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

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JoVE Journal

发育生物学

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

胎儿和成体神经干细胞衍生少突胶质细胞前体细胞培养物的高内涵筛选分化与成熟分析

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

Differentiating Human Neuronal Stem Cells into 3D Culture Using a Porous Scaffold

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Differentiating Human Neuronal Stem Cells into 3D Culture Using a Porous Scaffold