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1. Preparation and Maintenance of 3D Sphere Cocultures
<ol>
	<li>Dissociate hAstros from 3D aggregates in suspension.&#8203;
	<ol>
		<li>Gently dissociate human astrocyte (hAstro) aggregates with detachment solution when dark centers appear and remove spheres that spontaneously attach. Break spheres gently once a week to maintain sphere health and to avoid necrotic cores. 
		&#8203;NOTE: Do not exceed 5 min of treatment with detachment solution.</li>
	</ol>
	</li>
	<li>Dissociate hNeurons or iNeurons from a 2D monolayer.
	<ol>
		<li>Remove media from the 6-well plate and add 500 &#181;L of detachment solution to each well-containing monolayer culture of hNeuron (human neuron) or iNeurons (inducible neurons). Incubate at 37 &#176;C for 5 min. Gently add 2-3 mL DMEM/F12 basal medium to remove attached cells.</li>
		<li>Collect cells and media in a 15 mL conical tube and centrifuge at 300 x g for 1 min. Aspirate the supernatant, add 1 mL of fresh media, and pipette up and down gently with a 1,000 &#181;L micropipette to achieve a single-cell suspension.</li>
	</ol>
	</li>
	<li>Form 3D spheres using microwell culture plates.
	<ol>
		<li>Prepare the plate by adding 0.5 mL of anti-adherence rinsing solution to each well of the microwell plate. Centrifuge plate at 2,000 x g for 5 min. Aspirate rinsing solution, add 1 mL of DMEM/F12 basal medium to each well to wash, and aspirate again. 
		NOTE: Always ensure that the centrifuge is balanced during use.</li>
		<li>Count each cell type using a hemocytometer or automated cell counter. Add desired ratio of dissociated hAstros and hNeurons or iNeurons to each well in a total volume of 2 mL of neural medium (NM) + Y (include Dox if iNeurons are utilized). Centrifuge plate at 100 x g for 3 min and return to the incubator. 
		NOTE: 24-well microwell plates (see the Table of Materials) contain 300 microwells per well. Add a minimum of 6 x 105 cells (for spheres of 2 x 103 cells each) and a maximum of 6 x 106 cells (for spheres of 2 x 104 cells each) in 2 mL of media per well. For viable spheres of a specific density within this range, use a defined quantity of cells and divide by 300 to calculate the number of cells per sphere. Alternative sphere-forming methods and tools may also be utilized if desired. Follow the manufacturer&#39;s instructions for acceptable ranges of cell densities.</li>
		<li>Allow cells to self-assemble into densely packed spheres within microwell plates over the next 2 days (Figure 1A). Replace 50% of media if the culture is yellowing. 
		NOTE: Exchange only half media and pipette gently when removing and adding media in order not to disturb spheres. Spheres may lift up from microwells and fuse together with higher force.</li>
		<li>After 2 days, gently remove spheres from microwells with a 1,000 &#181;L micropipette (Figure 2D). Lightly apply force to the bottom of microwells with media to remove any additional adhered spheres. Let spheres settle in a 15 mL conical tube, aspirate the old media, and add fresh media.</li>
	</ol>
	</li>
	<li>Set up the spinner flask bioreactor system for forming 3D spheres.&#8203;
	<ol>
		<li>Clean the surface of a magnetic stir plate with 70% ethanol, and place it into a cell culture incubator. Autoclave individual spinner flasks to sterilize.</li>
		<li>Add spheres with a minimum of 50&#8211;60 mL media to each spinner flask (Figure 2D, inset). Place the flask on the magnetic stir plate set at 60 rpm. Culture spheres in spinner flasks until they are ready for data collection. 
		NOTE: A minimum of 3 weeks in culture is needed for synapse formation.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6 well plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td>Detachment solution</td>
		</tr>
		<tr>
			<td>AggreWell plate</td>
			<td>Stemcell Technologies</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>Stemcell Technologies</td>
			<td>7010</td>
			<td>Prevent cell adhesion to microwell plates</td>
		</tr>
		<tr>
			<td>Anti/anti</td>
			<td>Thermofisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>BrainPhys neuronal medium</td>
			<td>Stemcell Technologies</td>
			<td>5790</td>
			<td>Neurophysiological basal medium alternative</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>10565-042</td>
			<td>With GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>DMH-1</td>
			<td>Stemcell Technologies</td>
			<td>73634</td>
			<td>HAZARD: Toxic if swallowed. Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Lampire Biological Laboratories</td>
			<td>7332100</td>
			<td>Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer</td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride (Dox)</td>
			<td>Sigma</td>
			<td>D3072-1ml</td>
			<td>HAZARD: Toxic for pregnant women. Working concentration: 2 ug/mL</td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-2 (FGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Hemacytometer or automatic cell counter</td>
			<td>Life Technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.</td>
		</tr>
		<tr>
			<td>MEA 2100 System</td>
			<td>Multichannel Systems</td>
			<td>MEA2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Rho-Kinase Inhibitor Y27632- (Y)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Working concentration: 10 uM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemcell Technologies</td>
			<td>72234</td>
			<td>Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Spinner flasks</td>
			<td>Fisher Scientific</td>
			<td>4500-125</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-207</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating 3d co-culture spheres of astrocytes and neurons to induce synapse formation

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates a procedure for co-culturing the astrocytes and neurons to form 3D spheres. These 3D spheres provide a distinct platform for examining synapse formation and neural network integration, thereby serving as a potential model for research in developmental neuroscience, neurodegenerative diseases, and neuropharmacological interventions.

<img alt="Figure 1" src="/files/ftp_upload/22392/22392_Figure1.jpg" /> 
Figure 1: Systematic and reproducible generation of bioengineered 3D neural spheres. (A) Microwell (&#181;-well) culture plates are used to form 3D neural spheres of desired density and neuron-to-astrocyte ratios in a systematic and user-specified manner, yielding consistent size and shape. Images are shown 1 day after sphere formation. Scale bars = 200 &#181;m. (B) A range of starting cell densities (from 4 x 103 to 2 x 104 cells per microwell) produces spheres of varying sizes. In the absence of microwell plates, hPSCs combine to form significantly larger and nonuniform aggregates whose diameters surpass the limit of diffusion after a week (n = 6-14 spheres per group and time point). (C) iNeuron spheres cultured in a spinner flask at 80 rpm exhibited less fusion and thus were significantly smaller, more uniform, and exhibited greater circularity compared to those in stationary culture over a period of three weeks (n = 6-51 spheres per group and time point). Plots represent mean &#177; SD; * indicates significance (p &#60; 0.05) between groups, determined using two-tailed t-tests.
<img alt="Figure 2" src="/files/ftp_upload/22392/22392_Figure2.jpg" /> 
Figure 2: Stepwise depiction of the differentiation and formation of 3D neural spheres derived from hPSCs. (A) Timeline of key steps in the protocol. (B) Pure populations of neural cells can be generated from human induced pluripotent stem cells (hPSCs). For the generation of astrocyte progenitors (dotted green box). (C) Inducible neurons (iNeurons) generated from transgenic hPSCs via induced overexpression of neurogenin 2 demonstrate neuronal morphology on 2D ECM (day 7) and are positive for MAP2 (inset). (D) Spheres removed from microwell plates demonstrate consistent size for high-throughput screening. Spheres may be cultured in a spinner flask bioreactor (inset; see step 1.4) if desired to prevent fusion. Scale bar = 50 &#181;m (C, inset). Scale bars = 200 &#181;m (A, D).

Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation

1. Preparation and Maintenance of 3D Sphere Cocultures

	Dissociate hAstros from 3D aggregates in suspension.&#8203;
	
		Gently dissociate human astrocyte ...

Take a multiwell plate coated with a surfactant to reduce the surface tension of the well.
Add the desired ratio of astrocytes and neurons in a suitable culture medium.
Centrifuge to settle the cells and then incubate.
The low surface tension prevents cells from sticking to the well surface, causing their aggregation.
The cells adhere to each other through various membrane interactions, including adhesion molecules, forming a 3D coculture sphere.
Collect the spheres and transfer them to a tube.
Allow the spheres to settle. Remove the supernatant and add fresh culture medium.
Transfer the spheres to a spinner flask containing a suitable growth medium and incubate with constant spinning.
In the sphere, the neurons extend projections towards each other, forming a junction called a synapse, while the astrocyte extends towards the synapse, establishing a close association with the neurons.

generating-3d-co-culture-spheres-astrocytes-neurons-to-induce-synapse

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

65 Views. Source: Cvetkovic, C., et al. Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells. J. Vis. Exp. (2018). This video demonstrates a procedure for co-culturing the astrocytes and neurons to form 3D spheres. These 3D spheres provide a distinct platform for examining synapse formation and neural network integration, thereby serving as a potential model for research in developmental neuroscience, neurodegenerative diseases, and neuropharmacolo...

Video: Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation - Concept

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

JoVE Journal

Biochemistry

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as &#34;matrix&#34;), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

Developmental Biology

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation

Transcript

Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis