Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Our research aims to integrate in vitro and in sillico models to inform drug discovery and development at early stages of development. In a nutshell, we combine stem cell technology, organoids, and microfluidics to investigate drug-disease interactions, membrane integrity, and to predict drug absorption and drug metabolism. In our field, cutting-edge technologies include physiological base in silico models in combination with advanced in vitro systems, like micro-electrophysiological system, like organs-on-a-chip and organoids.

Additionally to this, 3D bioprinting is currently being explored to maximize the scalability of these more complex in vitro models. Current experimental challenges include enhancing tissue microenvironments and physiological fidelity of these in vitro models, minimizing the experimental variability and optimizing high-throughput capabilities. Also, establishing standardized protocols that are compliant with the regulatory standards for drug development.

With the goal of decreasing the experimental variability, better mimicking the tissue microenvironment, and adhere to the regulatory guidelines for human drug development, this protocol describe the optimization of an organoid generation and the maintenance of this organoid system using a xeno-free hydrogel system with a control composition and control mechanical properties. We are interested in developing microphysiological systems to model ADMETox in special populations, not well represented in clinical trials, like for example, people living with Down syndrome. Our lab is also working on bringing novel immunooncology-based treatment approaches to new settings across gastrointestinal and lung cancers.

To begin, place the tubes containing 25 milliliters of prepared cold advanced DMEM/F-12 media on ice. Add the thawed matrices to each tube. Mix the suspension together, ensuring that no clumps are formed.

Now pipette 250 microliters of the diluted matrices into each well of a 24-well plate. Tilt the plate to spread the coating solution evenly. After incubation, use a serological pipette to gently remove the excess solution without scratching the coated surfaces.

Immediately pipette 250 microliters of warm complete stem cell medium into each well. To coat the tissue culture plates with Matrix 4 xeno-free hydrogel, First, warm the hydrogel to room temperature. Add two volumes of the hydrogel to a tube containing one volume of 3x stem cells culture medium.

Pipette the solution up and down to mix it thoroughly. Transfer 250 microliters of the hydrogel into the wells of a 24-well tissue culture plate. Then tilt the plate to spread the hydrogel evenly.

After 10 to 15 minutes, the coated plates are now ready for the culture of induced pluripotent stem cells. To begin, pipette one to two milliliters of anti adherence rinsing solution to a 15-milliliter conical tube. Swirl the tube to coat its surface.

Next, pipette five milliliters of DPBS solution into the tube after removing the anti-adherent solution. Cap the coated tubes until needed. Pipette out any medium in a plate containing the human intestinal organoid domes.

With a one-milliliter pipette, add one milliliter of cold DMEM/F-12 medium to the dome directly to detach it from the plate. Pipette another milliliter of the medium to harvest any remaining organoids. Transfer the suspension to the coated 15-milliliter conical tubes.

Pipette the suspension up and down to disintegrate organoids until a uniform fragment suspension with the desired organoid size is created. Next, pipette out an aliquot of the suspension for counting. Place the remaining suspension on ice.

Draw an XY grid at the bottom of each well of a flat-bottomed 96-well plate. Pipette 50 microliters of DPBS into the wells. Transfer five microliters of the aliquot into each well.

Manually count the total number of organoids that are 50 to 200 micrometers in diameter. Then use the given equation to calculate the volume of the organoid suspension. After counting the organoids, remove the supernatant and the cloudy layer from the tube containing the remaining clumped suspension.

Then pipette two milliliters of cold DMEM/F-12 directly onto the pellet. Centrifuge the suspension at 200 g for five minutes at room temperature. For an animal-derived system, add 40 microliters of cold one animal-derived matrix to the organoid pellet.

Pipette the organoids up and down to distribute them into the matrix. Gently transfer a matrix organoid mixture into the center of a sterile prewarmed 24-well tissue culture plate. Incubate the plate at 37 degrees Celsius for 30 minutes to ensure dome gelation.

Next pipette, 0.5 milliliters of warm intestinal organoid growth media to the sides of the well without disturbing the dome before incubation. For the Matrix 4 xeno-free organoid system, add 50 microliters of 3x organoid growth media to the spheroid pellet. Then pipette 100 microliters of the selected Matrix 4 xeno-free organoid to the suspension.

Add 40 microliters of the hydrogel organoid mixture to the center of a 24-well tissue culture plate. Incubate the plate at 37 degrees Celsius for 30 minutes. After incubation, pipette 0.5 milliliters of organoid growth media along the sides of the well.

Then incubate the plate again at 37 degrees Celsius under 5%carbon dioxide supplementation and 95%humidity.