eoe sub articles

EoE Sub Articles

1. Making organoids from a single-cell suspension
<ol>
	<li>In an ice bucket or cold block, place the laminin-rich extracellular matrix (lrECM) and a small centrifuge tube. Place the appropriate amount of lrECM (16 &#181;L x X number of intended organoids) into the small centrifuge tube.</li>
	<li>Create a mixture of cells in a total volume (4 &#181;L x X number of intended organoids) containing 20,000 cells/organoid and add this to the small centrifuge tube with lrECM on ice.</li>
	<li>Carefully pipet...

generating an organoid culture from a brain tumor cell suspension

Source: Sundar, S. J. et al.&#160; <a href="https://app.jove.com/v/63745">Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture.</a> J. Vis. Exp. (2022).
This video describes the preparation and cultivation of brain tumor organoids from single-cell suspension. Using a laminin-rich extracellular matrix as a scaffold and culture media for nutrients, cells are facilitated to multiply and self-organize within the organoid. Consequently, ex vivo organoids are generated, mimicking cellular diversity and the microenvironment of brain tumors.

<img alt="Figure 1" src="/files/ftp_upload/22422/22422_Figure1.jpg" /> 
Figure 1 shows early organoid growth seen via light microscopy at 10x magnification. Figure 1A shows the migration and invasion of single cells through lrECM in the center view. The cells will continue to expand and &#39;colonize&#39; the lrECM, and they will appear more dense and eventually opaque by visual inspection. Figure 1B shows several mature organoids (at 7 weeks) without magnif...

Generating an Organoid Culture from a Brain Tumor Cell Suspension

1. Making organoids from a single-cell suspension

	In an ice bucket or cold block, place the laminin-rich extracellular matrix (lrECM) and a small ...

Begin with a microcentrifuge tube containing a laminin-rich extracellular matrix or lrECM. Laminin is a structural component present abundantly in the brain.
Keep the tube on ice to prevent IrECM polymerization.
Add brain tumor cells and mix thoroughly to ensure a uniform distribution of cells.
Next, transfer the mixture onto parafilm molds, forming droplets.
Incubate to facilitate lrECM polymerization to form a scaffold, mimicking the cells&#39; natural environment.
Now, flush the solidified organoids into a Petri dish containing a culture medium, then incubate.
The culture medium provides essential nutrients and growth factors, supporting cell division.
Within the organoid, cells self-organize by migrating and invading.
After incubation, tilt the plate to settle the organoids.
Carefully remove the excess medium and add fresh medium.
Incubate with gentle shaking.
This distributes the nutrients evenly and enhances organoid growth.

generating-an-organoid-culture-from-a-brain-tumor-cell-suspension

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Hepatocellular Carcinoma Tissue Dissociation for Organoid Culture

To begin the tissue dissociation, collect one to two cubic centimeters of hepatocellular carcinoma or HTC tissue from untreated patients. Then place the tissue in the tissue preservation solution and maintain it at four degrees Celsius until processing. Now, preheat the ultra-low attachment surface cell culture plates for one hour in an incubator set at 37 degrees Celsius.Thaw the basement membrane extract overnight at four degrees Celsius. Using surgical scissors, cut the tumor tissue into small pieces on a Petri dish inside a laminar flow hood. Transfer these pieces into a 15-milliliter conical tube and add five to 10 milliliters of basal medium.With a barotropic pipette, wash away as much blood as possible. Allow the mixture to settle for one to two minutes before removing some of the supernatant, including any blood cells and floating fat clots. Next, centrifuge the mixture at 300 G for five minutes at room temperature.Then carefully extract the supernatant and add five milliliters of pre-warmed digestion solution to the trimmed tissue. Place the tube at 37 degrees Celsius in a rotor for optimum digestion. After 30 minutes of initial digestion, use a microscope to look for small cell clusters.To stop the digestion, add five milliliters of cold basal medium to the tube. Then use a 100-micrometer cell filter to sieve into a new 50-milliliter tube. Fill up the tube with cold basal medium to reach a total volume of 50 milliliters.Next, centrifuge the cells at two G for 10 minutes at eight degrees Celsius. Once complete, carefully remove the supernatant liquid. Re-suspend the pellet in 50 milliliters of cold basal medium to obtain the cell pellet for organoid plating.

JoVE Journal

Cancer Research

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple and practical solution. Therefore, a more feasible cerebral organoid protocol is proposed in the present study. To solve the unavoidable inconvenience in medium change and organoid transfer in the early stage, the current research optimizes the operation technology by applying the engineering principle. A soft light lamp was adopted to laterally illuminate the embryoid body (EB) samples, allowing the EBs to be seen by the naked eye through the enhanced diffuse reflection effect. Using the principle of secondary flow generated by rotation, the organoids gather toward the center of the well, which facilitates the operation of medium change or organoid transfer. Compared to the dispersed cell, the embryoid body settles faster in the pipette. Using this phenomenon, most of the free cells and dead cell fragments can be effectively removed in a simple way, preventing EBs from incurring damage from centrifugation. This study facilitates the operation of cerebral organoid culture and helps to promote the application of brain organoids.

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

Cerebral organoids provide unprecedented opportunities for studying organ development and human disease pathology. Although great success has been achieved with cerebral organoid culture systems, there are still operational difficulties in applying this technology. The present protocol describes a cerebral organoid procedure that facilitates medium change and organoid transfer.

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple ...

Bioengineering

Organoid Culture from a Single Intestinal Stem Cell

To generate organoid from a single erythropoietin-producing hepatocellular receptor B2, or FEB2-positive intestinal stem cell, carry out fluorescence-activated cell sorting based on the FEB2 expression, and divide the cells obtained into four groups, high, medium, low and negative. After collecting the FEB2 high cell pellet with centrifugation at 390G for three minutes at four degree Celsius, embed the single sorted FEB2 high cells in the ECM by pipetting, then seed the cells on a 24-well plate at 100 singlets per 40 microliters of ECM per well. Incubate the 24-well plate for 15 minutes at 37 degrees Celsius and 5%carbon dioxide for the polymerization of the ECM.Next, cover the ECM with 500 microliters of culture medium containing 10 micromolar row-associated kinase, or rock inhibitor for the first two days to maintain the FEB2 high cells. Manually inspect the cells using an inverted microscope at 40X magnification and observe viable organoids with spheroid formation and crypt protrusion. Self-organizing crypt villa structures reminiscent of the normal small intestine were recreated from single FEB2 high cells.By day five of culture, spheroid-like structures formed, and from day seven to day nine, evagination of the spots to form crypts occurred.

Generating an Organoid Culture from a Brain Tumor Cell Suspension

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