The authors acknowledge previous training and general recommendations regarding starting hiPSC and organoid work from Drs. Christina Pacak, Silveli Susuki-Hatano, and Russell D&#39;Souza. They thank Dr. Chelsey Simmons for her guidance in using hydrogel systems for in vitro cell culture work. Also, the authors would like to thank Drs. Christine Rodriguez and Thomas Allison from STEMCELL Technologies for their guidance on hiPSC culture. The authors also thank TheWell Bioscience for covering the publication costs.

1. hiPSC maintenance
CAUTION: All work is done in a Biosafety Cabinet (BSC) following standard aseptic techniques. Must follow OSHA safety standards for laboratories, including proper use of personal protective equipment such as lab coats, gloves, and goggles.
<ol>
	<li>Preparation of matrices aliquots and cell culture media
	<ol>
		<li>For commercially available animal-derived basement membranes (BMs; Matrix 1-AB, Matrix 2-AB, Matrix 3-AB), prepare aliquots of working volum...

Matrix Comparison for Efficient Human Intestinal Organoid Generation from iPSCs

<ol>
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Selecting the optimal microenvironment for stem cell and organoid work is a pivotal early step when using these platforms for a wide range of applications. Our representative results show that Matrix 4-XFO3, in combination with a higher concentration of growth factors, leads to larger organoids, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation using these systems. It has been previously shown that the unique characteristics of the extracellular matrix (ECM) a...

The extracellular matrix (ECM) is a dynamic and multifunctional component of tissues that plays a central role in regulating cell behavior and development. As a complex network, it provides structural support, cell adhesive ligands1, and storage of growth factors and cytokines that regulate cell signaling. For example, during wound healing, the ECM serves as a scaffold for migrating cells and as a reservoir of growth factors involved in tissue repair2. Similarly, dysregulation in the ECM can lead to an increase in the severity of various diseases such as fibrosis and cancer3,4. During embryonic development, the ECM guides tissue morphogenesis. For example, in the development of the heart, ECM components play a role in creating the correct architecture and function of the heart tissue5. Over a decade of research has shown that the stiffness of the microenvironment alone6,7 can control stem cell lineage specification. Therefore, it is not surprising that during in vitro cell differentiation, ECM influences stem cell fate by providing signals for differentiation.
Organoids can be generated from induced pluripotent stem cells (iPSCs). Starting with a properly characterized iPSC line is required to generate organoids successfully. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation and organoid generation. Moreover, recent research has emphasized the significance of the composition of the extracellular matrix (ECM), interactions between cells and the ECM8, as well as mechanical and geometrical cues9,10,11 in the context of organoid expansion and differentiation12. Advancing organoid technology by improving reproducibility will involve incorporating tissue-specific physical and chemical cues.
Organoids aim to recapitulate the native tissue within a physiologically similar microenvironment. Choosing an ECM system that closely mimics the native tissue ECM is crucial for achieving physiological relevance regarding cell behavior, function, and response to stimuli13. The choice of ECM components can influence the differentiation of stem cells into specific cell types within the organoid. Different ECM proteins and their combinations can provide cues that guide cell fate14. For example, studies have shown that using specific ECM components can promote the differentiation of intestinal stem cells into mature intestinal cell types, resulting in physiologically relevant intestinal organoids15. While organoids are a valuable tool during disease modeling and drug testing, selecting an appropriate ECM system is pivotal to this application. An appropriate ECM system can enhance the accuracy of disease modeling by creating a microenvironment that resembles the affected tissue16. Furthermore, tissue-specific ECM can help generate organoids that better recapitulate disease-associated phenotypes and drug responses17. Optimizing the ECM system used in organoid differentiation is critical for achieving desired differentiation outcomes.
Commercially available basement membrane systems derived from animal ECM sources (e.g., Matrigel, Cultrex) and xeno-free hydrogel (e.g., VitroGel) are widely used in iPSC and organoid research. Companies that commercialize them and researchers that use them have laid out many instructions for their specific products and applications over the years. Many of these instructions served as a guide for the generation of this protocol. Furthermore, the benefits and setbacks associated with their intrinsic properties have been individually noted by many18,19,20,21. However, there is no systematic workflow to guide the selection of optimal systems for iPSC and organoid work. Here, a workflow to systematically evaluate the equivalency of ECM systems from various sources for iPSC and organoid work is provided. This is a comparative study of the maintenance of two different human iPSC lines (hiPSC) and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF). For organoid culture, four versions of the xeno-free matrix VitroGel that were previously optimized for organoid culture were used: ORGANOID 1 (Matrix 4-O1), ORGANOID 2 (Matrix 4-O2), ORGANOID 3 (Matrix 4-O3), ORGANOID 4 (Matrix 4-O4). Also, animal-derived matrices optimized for organoids were used: Matrigel High Concentration (Matrix 1-ABO) and Cultrex Type 2 (Matrix 3-ABO). Commercially available stem cell culture media (mTeSR Plus) and organoid differentiation kit (STEMdiff intestinal organoid kit) were used. This protocol combines the individual instructions from the products&#39; manufacturers with lab experiences to guide the reader toward a successful optimization of ECM for their specific iPSC and organoid work. Altogether, this protocol and representative results emphasize the importance of selecting the optimal microenvironment for stem cell work and organoid differentiation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-Well Plate (Culture treated, sterile)</td>
			<td>Falcon</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C water bath</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate&#160;</td>
			<td>Fisher Scientific</td>
			<td>FB012931</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Life Technologies</td>
			<td>12634</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-adherence Rinsing Solutio</td>
			<td>STEMCELL Technologies</td>
			<td>7010</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet (BSC)</td>
			<td>Labconco&#160;</td>
			<td>Logic</td>
			<td></td>
		</tr>
		<tr>
			<td>Brightfield Microscope</td>
			<td>Echo Rebel</td>
			<td>REB-01-E2</td>
			<td></td>
		</tr>
		<tr>
			<td>BXS0116</td>
			<td>ATCC</td>
			<td>ACS-1030</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge with temperature control (4 &#176;C capabilities)</td>
			<td>ThermoScientific</td>
			<td>75002441</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes, 15 mL, sterile</td>
			<td>Thermo Fisher Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes, 50 mL, sterile</td>
			<td>Thermo Fisher Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex RGF BME, Type 2</td>
			<td>Bio-techne</td>
			<td>3533-005-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Stem Cell Qualified RGF BME&#160;</td>
			<td>Bio-techne</td>
			<td>3434-010-02</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS (Without Ca++ and Mg++)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>GeltrexLDEV-Free, hESC-Qualified Reduce Growth Factor</td>
			<td>Gibco&#160;</td>
			<td>A14133-02</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fischer Scientific</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Guava Muse Cell Analyzer or another flow cytometry equipment (optional)</td>
			<td>Luminex</td>
			<td>0500-3115</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer solution</td>
			<td>Thermo Fischer Scientific</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Heralcell Vios Cell culture incubator (37 &#176;C, 5% CO2)</td>
			<td>Thermo Scientific&#160;</td>
			<td>51033775</td>
			<td></td>
		</tr>
		<tr>
			<td>JMP Software</td>
			<td>SAS Institute</td>
			<td>JMP 16</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks, Inc</td>
			<td>R2022b</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix LDEV free</td>
			<td>Corning&#160;</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix High Concentration (HC), Growth Factor Reduced (GFR) LDEV-free</td>
			<td>Corning&#160;</td>
			<td>354263</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR Plus Medium</td>
			<td>STEMCELL Technologies</td>
			<td>100-0276</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon Delta surface treated 24-well plate</td>
			<td>Thermo Scientific</td>
			<td>144530</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-human CD326 (EpCAM)</td>
			<td>BD Pharmingen</td>
			<td>566841</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-human CDX2&#160;</td>
			<td>BD Pharmingen</td>
			<td>563428</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-human FOXA2</td>
			<td>BD Pharmingen</td>
			<td>561589</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP-Cy 5.5 Mouse Anti-human SSEA4&#160;</td>
			<td>BD Pharmingen</td>
			<td>561565</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>STEMCELL</td>
			<td>5872</td>
			<td></td>
		</tr>
		<tr>
			<td>SCTi003-A</td>
			<td>STEMCELL Technologies</td>
			<td>200-0510</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes (10 mL)&#160;</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes (5 mL)&#160;</td>
			<td>Fisher Scientific</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Intestinal Organoid Growth Medium</td>
			<td>STEMCELL Technologies</td>
			<td>5145</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Intestinal Organoid Kit</td>
			<td>STEMCELL Technologies</td>
			<td>5140</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrogel Hydrogel Matrix</td>
			<td>TheWell Bioscience</td>
			<td>VHM01</td>
			<td></td>
		</tr>
		<tr>
			<td>VitroGel ORGANOID Discovery Kit</td>
			<td>TheWell Bioscience</td>
			<td>VHM04-K</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparative study of basement-membrane matrices for human stem cell maintenance and intestinal organoid generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

Author Spotlight: Advancing Organoid Generation for Drug Development Using iPSCs

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

Following this protocol, commercially available basement membranes and a xeno-free hydrogel system were successfully utilized to cultivate hiPSC cells and differentiate them into hIO. The main objective of these experiments was to systematically evaluate the equivalency of matrices from various sources for hiPSC and hIO work. The first section of this protocol focused on the maintenance and characterization of a healthy iPSC culture that yields an efficient intestinal organoid generation. The process of coating the cultu...

Read this Scientific Article Protocol about Author Spotlight: Advancing Organoid Generation for Drug Development Using iPSC at JoVE.com

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...