a technique to optimize cerebral organoid culture using lateral soft light illumination

A Technique to Optimize Cerebral Organoid Culture Using Lateral Soft Light Illumination

Use a transparent acrylic board with a thickness of 0.3 to 0.5 centimeters in the size of A5 paper, and paste white pads on the front and back of the acrylic plate. Next, install a row of LED white lights on the edge of the plate so that the lights can enter from the side of the acrylic plate and then shoot out in parallel.
Now, prepare a new six-well low-adhesion plate, and add 2 milliliters of EB formation medium to each well. After removing the EBs together with the medium, using 1000-microliters wide-bore pipette tip, transfer approximately 100 EBs per well to the 6-well low-adhesion plate.
To replace the EB formation medium every day with the same volume of fresh medium, induce a swirl flow by rotating the dish along a circular orbit. The EBs or organoids converge to the center of the well due to the secondary flow generated through rotation. Aspirate the old medium by pipetting to the edge of the well slowly. Do not suck too hard, otherwise, the EBs will be removed together. Then, add fresh media to resuspend the EBs.
For neural induction, prepare a new 6-well low-adhesion plate with 3 milliliters of neural induction medium per well. Turn on the lateral soft light and turn off other indoor light sources. Now, transfer the EBs to the 6-well plate with added neural induction medium by using a wide-mouth pipette tip to suck both the EBs and the culture medium. Then, hold the pipette upright. The EBs will gradually sink under gravity, and converge towards the mouth of the pipette tip.
As the mouth of the pipette tip touches the liquid surface again, and due to liquid surface tension, the EBs quickly sink into the medium. Incubate the samples at 37 degrees Celsius and 5% carbon dioxide for 24 hours.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of the soft light lamp (day 1)
<ol>
	<li>Use a transparent acrylic board with a thickness of 0.3-0.5 cm in the size of A5 paper. Paste white pads on the front and back of the acrylic plate.</li>
	<li>Install a row of LED white lights on the edge of the plate so that the lights can enter from the side of the acrylic plate and then shoot out in parallel (Figure 1A-F, Figure 2B). 
	NOTE: As the diameters of EBs in the early stage are approximately 200 &#181;m to 300 &#181;m, it is difficult to observe them clearly with the naked eye under the fluorescent lamp of clean benches. In contrast, due to the enhancement of diffuse reflection, we can detect the EBs clearly by using laterally illuminated soft light (Figure 2B, C). A 6-well plate is recommended for EB cultures in subsequent experiments. However, to better show the visual effect of soft light, dishes are sometimes used to take pictures and videos in this study instead of plates, so please do not misunderstand. The laterally illuminated soft light lamp can also be used for the 6-well plate.</li>
</ol>
2. EB transfer and medium replacement (days 2-5)
<ol>
	<li>Prepare a new 6-well low adhesion plate and add 2 mL of embryoid body (EB)-formation medium to each well.</li>
	<li>Remove the EBs together with the medium with a 1000 &#181;L wide-bore pipette tip (see&#160;Table of Materials) and transfer them to the 6-well low adhesion plate (~100 EBs/well). 
	NOTE: The operation process adopts a soft light lamp (mentioned in step 1) to make the EBs easier to observe. Turn off other indoor light sources to improve the visual effect of the soft light.</li>
	<li>Replace with the same volume of fresh EB-formation medium every day. Use the secondary flow to gather the EBs to the center and change the medium.
	<ol>
		<li>Aspirate the old medium by pipetting to the edge of the well slowly. Do not suck too hard; otherwise, the EBs will be removed together. Then, add fresh medium to resuspend the EBs. 
		NOTE: The principle secondary flow (Figure 2D). Induce a swirl flow by rotating the dish along a circular orbit. Due to the swirl flow, a secondary flow is induced and directed toward the center. The EBs or organoids converge to the center of the well due to the secondary flow generated through rotation, after which medium change or embryoid transfer can be executed readily.</li>
	</ol>
	</li>
</ol>
3. Neural induction (days 5-7)
<ol>
	<li>Prepare a new 6-well plate with low adhesion and add 3 mL of Neural induction medium (Table 1) to each well.</li>
	<li>Turn on the lateral soft light (mentioned in step 1) and turn off other indoor light sources.</li>
	<li>Transfer the EBs to the 6-well plate with added Neural induction medium (~100 EBs/well). Add as little as possible of the original medium to the new well. 
	NOTE: Introduce simple skills of organoid transfer. Naturally, under gravity and with a relatively higher density than the medium, the resuspended EBs will gradually sink by applying the operation shown in&#160;Figure 2E. Hence, the EBs can be conveniently transferred. Since, compared to EBs, free cells and dead cell fragments sink more slowly, most of the free cells and dead cell fragments can thus be removed through this sedimentation method (Figure 2F).</li>
	<li>Incubate the samples at 37 &#176;C and 5% CO2&#160;for 24 h. 
	NOTE: Under the microscope, the diameter of the EBs was approximately 500 &#181;m, and the edge was translucent, indicating that a neuroepithelial layer formed.</li>
</ol>
Table 1. Composition of the Neural induction medium
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume </td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>97 mL</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Glutamax supplement</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Final concentration 1 &#956;g/mL</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m Filter</td>
			<td>NEST Biotechnology, China</td>
			<td>331001</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L wide-bore pipette tip</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>9405163</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well low adhesion plate</td>
			<td>NEST Biotechnology, China</td>
			<td>703011</td>
			<td>It is used for EBs suspension cultures</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, Germany</td>
			<td>5810 R</td>
			<td>It can be used for centrifugation of various types of centrifuge tubes, reagent bottles and working plates.</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck, Germany</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft light lamp</td>
			<td>NUT</td>
			<td>NUT</td>
			<td>A simple self made device, refer to supplementary figure 2 for preparation.</td>
		</tr>
	</tbody>
</table>

Source: Chen, B. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/63989">Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination.</a>&#160;J. Vis. Exp.&#160;(2022).
This video showcases the utilization of a lateral soft light illumination technique to improve the naked-eye visualization of embryoid bodies, thereby facilitating their manipulation and transfer during cerebral organoid culture. It elucidates the setup of soft light illumination, methods for media changes and embryoid body transfer, as well as the procedure for inducing neural differentiation in embryoid bodies for cerebral organoid development.

<img alt="Figure 1" src="/files/ftp_upload/22590/22590_Figure1.jpg" /> 
Figure 1: Preparation of the soft light lamp.&#160;(A) The power supply and the LED lights were installed on one side of the acrylic board (as shown in the red dotted box). The LED soft light lamp&#39;s scattering wavelength is 450-470 nm, luminous flux is 1300-1800 lm, and color rendering index is 75-85 Ra. (B) It was checked whether the LED bulbs could light no...

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of the soft light ...

Take a lateral soft light illumination setup containing an acrylic board fitted with white pads and white LED lights installed on one edge.
Position a low-adhesion multi-well plate containing embryoid body formation medium in front.&#160;
Add the embryoid bodies, or EBs to the plate.
The LED light enters from the side of the acrylic board and emits parallel beams, laterally illuminating the EBs and enabling naked-eye visualization.
Rotate the plate to induce a swirl flow, converging the EBs to the center.
Replace the spent medium with fresh EB formation medium.
Collect the EBs using a wide-bore pipette tip.
Hold the pipette upright to allow the EBs to settle by gravity.
Transfer the EBs to another low-adhesion plate containing a neural induction medium.
Touch the tip to the medium. The EBs sink due to liquid surface tension.
Specific factors in the medium initiate neural differentiation in the EBs, forming a neuroepithelial layer.

11 Views. Методика оптимизации культуры церебральных органоидов с использованием бокового мягкого светового освещения

Video: Методика оптимизации культуры церебральных органоидов с использованием бокового мягкого светового освещения - Experiment

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Анализ ретиноевой кислоты-индуцированной Neural дифференциации эмбриональных стволовых клеток мыши в двух и трех-мерных эмбриональных телец

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

JoVE Journal

Developmental Biology

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

Разработка HiPSC-продуцированных сывороточных свободных эмбриоидных тел для опроса трехмерных культур стволовых клеток с использованием физиологически релевантных анализов

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

Моделирования Зика вирус развивающегося мозга человека в пробирке с помощью стволовых клеток получены мозгового Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

A Technique to Optimize Cerebral Organoid Culture Using Lateral Soft Light Illumination

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A Technique to Optimize Cerebral Organoid Culture Using Lateral Soft Light Illumination