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.

Matrix Comparison for Efficient Human Intestinal Organoid Generation from iPSCs

<ol>
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Dr. John Huang is founder &amp; CEO at TheWell Bioscience.

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) are closely intertwined with the identity of the cells within the organ12. Because cells sense external forces via integrins and other receptors, the mechanical characteristics of the ECM profoundly influence stem cells, and in response, stem cells can generate internal forces12. Interactions between the stem cells and their microenvironment are key in initiating diverse signaling pathways12. Additionally, it has been shown that the ECM can serve as a reservoir for storing and releasing growth factors and other signaling molecules that impact cell behavior. Hence, our representative results and decades of research emphasize the importance of considering biochemical and physical aspects of the native cell microenvironment and tissue properties when selecting matrix systems for in vitro investigations29,30.
Several critical steps and recommendations were explained through the protocol to advise the users of potential issues and ways to avoid them. Nevertheless, two critical steps are presented in these methods that are pivotal for successfully implementing this protocol. First, checking for specific markers associated with the stem cell population is crucial to determining the optimal coating system to maintain the stem cell population ready for efficient controlled differentiation. Similarly, confirming the expression of markers associated with each differentiation step is essential to deciding when to proceed to the next stage. This protocol provides a general guideline regarding the minimum time required at each stage. Nevertheless, the optimal time at each stage will vary for different cell lines and experimental conditions (e.g., genotype, stiffness). Therefore, characterizing multiple stage-specific markers is the best approach to improve the efficiency of hIO generation.
The second critical step was incorporating a higher concentration of growth factors when using the xeno-free Matrix 4-XF system, which is of utmost importance. One frequently highlighted limitation of xeno-free biomaterial systems is the lack of growth factors31. However, this weakness can become a strength: full control of growth factors in the systems to improve reproducibility. While this protocol describes using a commercially available media and growth factor supplementation kit, the same rationale can be used when making the complete media in-house with individual components. The specific ratio hydrogel/media guides how much higher needs to be the concentration of growth factors; for example, a 2:1 ratio might require 3x concentration of growth factors to end up with a concentration representative of the standard complete media used. The full control of growth factors present in the system is beneficial to answering mechanistic questions and avoiding regulatory issues for many applications.
The image processing steps described in step 3 are limited by the quality of the images and the type of images taken. It might require refinement to suit the specific requirements of each image type and quality. However, the overall steps of the process and a sample of MATLAB code of the basic framework for organoid size analysis from brightfield images help to define a workflow that can be further optimized and expanded. Furthermore, access to a brightfield microscope is more common than other types of microscopes. While lack of access and familiarity with MATLAB might limit the use of this code, the general steps of the process are compatible with open-source software such as Fiji by Image J. Organoid size alone is not a recommended metric to assess maturation; it is encouraged to follow with an evaluation of specific markers representative of the target organ; some of these markers for intestinal tissue are included in Table 4. Figure 6 shows an example of immunofluorescence staining of whole organoids with the epithelial cell adhesion molecule (EpCAM), which is localized to the exterior surface (basolateral) of the organoids. While EpCAM was visualized in the organoids cultured in all the ECM systems, there were differences in the size of these organoids. Thus, information about organoid size helps to optimize the culturing conditions. Furthermore, in drug response studies, organoid size serves as a metric of the response to the tested compound; changes in size can reflect the effectiveness or toxicity of the tested compounds. Importantly, generating multiple batches of organoids with standardized size is a current challenge in the field, but it is an important metric of experimental reproducibility so results can be compared across different experiments or laboratories. Altogether, an automated workflow that allows the extraction of important information about organoids from brightfield images is a beneficial tool for many researchers.
Both systems, animal-derived matrices, and xeno-free hydrogels, have their advantages and disadvantages. Matrix 2-AB leading to fewer spheroid releases during the MH stage than the other animal-derived matrices could result from lot-to-lot variations, a widely known characteristic of commercially available animal-derived matrices. It is important to note the concentration of the commercially available ECM systems varies; for example, the dilution factors in the product specification sheets were used to prepare the coating solutions which might lead to slightly different concentrations in the case of the animal-derived matrices. These differences in protein concentration can significantly impact differentiation leading to variation in differentiation efficiency when using the same product from different lots. To select between these systems, consider factors such as specific experimental goals, ethical considerations, and regulatory requirements. For example, in cases of comparing in vitro experimental results with a mouse animal model to answer tumor and cancer biology questions, using matrices derived from the Engelbreth-Holm Swarm EHS mouse can be a great choice. Also, because products such as Matrigel have been used for decades, there are many protocols, data, and references. However, using well-defined xeno-free matrices that allow the customization of physical and chemical properties might be a more suitable option when the research question is associated with human diseases or therapies. Also, most xeno-free hydrogel systems are designed to meet regulatory standards, which makes them a suitable choice for research that requires compliance with good manufacturing practices (GMP) or other regulatory guidelines.
In summary, while animal-derived matrices have been widely used for iPSC maintenance and organoid generation due to their biocompatibility, xeno-free hydrogel systems offer advantages in terms of reproducibility, cost-effectiveness, regulatory compliance, and ethical considerations. The choice between the two depends on the specific needs of the research or clinical application. Xeno-free hydrogel systems are gaining popularity as a more versatile and sustainable alternative that can support the utilization of organoids and microphysiological systems for the pre-clinical shortlist of therapeutic candidates. This application can significantly improve early drug development and help tackle current clinical translation challenges in the pharmaceutical industry.

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><tbody><tr><td>24-Well Plate (Culture treated, sterile)</td><td>Falcon</td><td>353504</td><td></td></tr><tr><td>37 &amp;deg;C water bath</td><td>VWR</td><td></td><td></td></tr><tr><td>96-well plate&amp;nbsp;</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&amp;nbsp;</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 &amp;deg;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&amp;nbsp;</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&amp;nbsp;</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 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Thermo Scientific&amp;nbsp;</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&amp;nbsp;</td><td>356231</td><td></td></tr><tr><td>Matrigel Matrix High Concentration (HC), Growth Factor Reduced (GFR) LDEV-free</td><td>Corning&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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)&amp;nbsp;</td><td>Fisher Scientific</td><td>13-678-11E</td><td></td></tr><tr><td>Serological pipettes (5 mL)&amp;nbsp;</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.

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 culture ware with animal-derived matrices Matrix 1-AB, Matrix 2-AB, and Matrix 3-AB was described and compared with a xeno-free hydrogel system Matrix 4-XF.
As shown in Figure 3A, the hiPSCs BXS 0116 (CD34+ derived) show comparable morphology when cultured on the three animal-derived matrices. Nevertheless, comparing the cell line BXS 0116 with SCTi003-A (PBMC derived) cultured on Matrix 1-AB-coated wells emphasizes that intrinsic differences between cell lines (shown in Table 6) can lead to variation regarding cell proliferation, differentiation, and morphology. For example, while it took 3 days to generate DE for the SCTi003-A cell lines, it took 5 days for the BXS0116 line. Moreover, as shown in Figure 3B-C, culturing hiPSCs in Matrix 4-XF hydrogel systems lead to colonies forming in a partially embedded 3D colony that is drastically different from the flat morphology of hiPSC culture on plates coated with Matrix 1-AB or equivalent systems. Also, one of the key aspects to note from these experiments is that making a xeno-free hydrogel system using 3x growth factors can considerably improve cell viability and expression of stem cell markers. Altogether, hiPSC cultures on animal-derived matrices and xeno-free Matrix 4-XF made with 3x growth factors concentration led to an equivalent expression of SSEA-4 (stem cell marker)22; however, the hydrogel system promotes the formation of partially embedded 3D colonies.
The second section of this protocol focuses on the differentiation of hiPSCs to generate hIO. Here, a commercially available kit developed based on Spence et al.23 was used to differentiate into definite endoderm (DE), then midgut/hindgut (MH), and finally collect spheroid to mature into IO (Figure 4C). For an efficient generation of IO, examining that the hiPSC culture has minimal spontaneous differentiation before starting the differentiation protocol is crucial. As shown in Figure 4A, the lower the expression of stem cell markers such as SSEA-4 when starting the DE differentiation, the less efficient the process will be at every stage (i.e., lower expression of MH markers) and subsequent poor generation of hIO. Utilizing a compact benchtop flow cytometry device facilitated quick access to flow cytometry results and deciding about the quality of differentiation before moving to the next stage. A peculiar observation was that Matrix 2-AB-maintained hiPSC resulted in fewer spheroid releases during midgut/hindgut stage compared to the other animal-derived systems. Another key aspect to note is that because colonies of hiPSC maintained in Matrix 4-XF already had a 3D structure at the beginning of the differentiation, these systems led to larger spheroids.
Spheroids were embedded into Matrix 1-ABO, Matrix 3-ABO, and Matrix 4-XFO1- Matrix 4-XFO4 and were matured into hIO using a kit. After maturing spheroids for 7 days, they were passaged and embedded into their respective matrices systems. The growth of the organoid on each system was tracked for 7 days from brightfield images. While the culture started with relatively similar-sized organoids, after 5 days, hIO size varied as a function of the matrix in which they were embedded (Figure 5). Of the four Matrix 4-XFO, the formulation that resulted in larger organoids was Matrix 4-XFO3. The Matrix 4-XFO systems are formulated with different ligands and mechanical properties to fit a variety of applications24. Specifically, Matrix 4-XFO3 stiffness25 is closer to human gut stiffness26 than the other Matrix 4-XFO formulations, which supports recent findings about the role of ECM mechanical properties on organoid expansion12,27,28. Our representative results indicate that Matrix 1-ABO and Matrix 3-ABO equally facilitate the growth and expansion of intestinal organoids.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66277/66277fig03.jpg" /> 
Figure 3: Selecting matrix system for iPSC maintenance. (A) Representative images of two cell lines grown on culture ware coating with the three different animal-derived matrix systems. (B) Representative images comparing the use of Matrix 4-XF with 1x vs. 3x growth factor supplementation show that increasing growth factor supplementations improves the viability&#160;of hiPSC on Matrix 4-XF hydrogel. (C) Flow cytometry comparison of SSEA-4 expression of Matrix 1-AB, Matrix 3-AB, Matrix 4-XF (3X) shows equivalent expression while Matrix 4-XF (1X) led to lower expression of SSEA-4. Bars&#160;represent mean &#177; SD of n = 5; two-way ANOVA, and post-hoc testing with nonparametric Wilcoxon method was used for significance testing. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/66277fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66277/66277fig04.jpg" /> 
Figure 4: Differentiating iPSC into definite endoderm (DE) and midgut/hindgut (MH). (A) Representative images of iPSC differentiation into DE starting with different levels of SSEA4 marker highlight the importance of starting differentiation with a hiPSC population with high expression of stem cell markers. The red arrows point to areas of spontaneous differentiation prior to starting DE. (B) Example of flow cytometry analysis of the stem cell marker SSEA4 expression before starting differentiation and DE marker FOXA2 after 3 days of DE differentiation for a poor DE differentiation (left) and a good differentiation (right). Each graph represents the analysis of cells extracted from 1; for statistical analysis, it is recommended to average the results from at least 3 flow cytometry runs. (C) Representative images of iPSC differentiation into hIO on all matrices studied. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/66277fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66277/66277fig05.jpg" /> 
Figure 5: Selecting matrix system for spheroid embedding and hIO maturation. (A) Sample images of maturation of hIO embedded in Matrix 4-XFO 1-4. The elastic modulus of the Matrix 4-XFO systems is around 50-300 Pa, with the highest for Matrix 4-XFO3 &#62; Matrix 4-XFO4 &#62; Matrix 4-XFO2 &#62; Matrix 4-XFO1. Matrix 1-ABO and Matrix 3-ABO stiffness range from 440-800 Pa depending on the batch. (B) Sample images of the maturation of hIO were embedded in Matrix 1-ABO, Matrix 3-ABO, and Matrix 4-XFO3 on the day of embedding (Day 0) and after 1 week of culture (Day 7). (C) Data showing size comparison of hIO matured on the different matrix systems. Bars represent mean &#177; SD of n = 7; two-way ANOVA, and post-hoc testing with nonparametric Wilcoxon method was used for significance testing. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/66277fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66277/66277fig06.jpg" /> 
Figure 6: Representative immunofluorescence images of intestinal organoids grown in the different matrices confirm the epithelial cell population. Whole intestinal organoids grown in the different matrices were fixed and stained with epithelial cell adhesion molecule (EpCAM) (green) and counterstained for cell nuclei DAPI (blue). All the organoids show EpCAM-positive cells, confirming the epithelial cell population localized on the exterior surface of the organoids. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/66277fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Summary of aliquot preparation, storage, and dilution of matrices used to coat culture ware used for iPSC. * For accurate dilution factor, check the Dilution factor recommended and/or protein concentrations in the Certificate of Analysis of each lot. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/Table 1_Summary of aliquot preparation_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Reference table of the volume of coating solutions per surface area of culture ware and mTeSR Plus Medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/Table 2_Volume of coating solutions_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Reference table of the volume of Matrix 4-XF precursor solutions per surface area of culture ware and complete stem cell medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/Table 3_Reference table of the volume of Matrix4-XF_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: List of common markers recommended to characterize hiPSC and hIO during the process. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/Table 4_List of common markers_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Summary of media needed during hiPSC differentiation into Intestinal Organoids. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/Table 5_Summary of media needed during hiPSC_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 6: Specific details about the two hiPSC lines used in this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/Table 6_iPSC lines information_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: General MATLAB Code to modify according to specific image type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66277/Code image Processing_RE.zip" target="_blank">Please click here to download this File.</a>

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Author Spotlight: Advancing Organoid Generation for Drug Development Using iPSCs

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.

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) ...

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1.1K Views.  University of Florida. Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM) 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.

Video: Author Spotlight: Advancing Organoid Generation for Drug Development Using iPSCs

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Cancer Research

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Developmental Biology

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Regional specification in the intestine is driven by multiple developmental pathways, including the bone morphogenetic protein (BMP) pathway. Based on normal regional specification, a method to generate human colonic organoids (HCOs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hES) and induced pluripotent stem cells (iPSCs), was developed. A three-day induction of BMP signaling sufficiently patterns mid/hindgut tube cultures into special AT-rich sequence-binding protein 2 (SATB2)-expressing HCOs containing all of the main epithelial cell types present in human colon as well as co-developing mesenchymal cells. Omission of BMP (or addition of the BMP inhibitor NOGGIN) during this critical patterning period resulted in the formation of human intestinal organoids (HIOs). HIOs and HCOs morphologically and molecularly resemble human developing small intestine and colon, respectively. Despite the utility of HIOs and HCOs for studying human intestinal development, the generation of HIOs and HCOs is challenging. This paper presents methods for generating, maintaining, and characterizing HIOs and HCOs. In addition, the critical steps in the protocol and troubleshooting recommendations are provided.

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing ...

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Biology

Developing 2D Monolayer Cultures from Bovine Intestinal Organoids

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

Author Spotlight: Development and Characterization of 2D Intestinal Monolayer Models from Bovine Organoids for Pathogen Interaction Studies

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...

Medicine

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of the human intestinal epithelium, in which they exhibit crypt/villus-like structures. Here, we describe the generation of hiPSC-derived intestinal organoids by the stepwise differentiation of hiPSCs into definitive endoderm, which is then posteriorized to form hindgut epithelium before being transferred into 3D culture conditions. The 3D culture environment consists of extracellular matrix (ECM) (e.g., Matrigel or other compatible ECM) supplemented with SB202190, A83-01, Gastrin, Noggin, EGF, R-spondin-1 and CHIR99021. Organoids undergo passaging every 7 days, where they are mechanically disrupted before transfer to fresh extracellular matrix and allowed to expand. QPCR and immunocytochemistry confirm that hiPSC-derived intestinal organoids contain mature intestinal epithelial cell types including goblet cells, Paneth cells and enterocytes. Additionally, organoids show evidence of polarization by expression of villin localized on the apical surface of epithelial cells.
The resulting organoids can be used to model human intestinal development as well as numerous human intestinal diseases including inflammatory bowel disease. To model intestinal inflammation, organoids can be exposed to inflammatory mediators such as TNF-&#945;, TGF-&#946;, and bacterial LPS. Organoids exposed to proinflammatory cytokines display an inflammatory and fibrotic phenotype in response. Pairing of healthy versus hiPSCs derived from patients with IBD may be useful in understanding mechanisms driving IBD. This may reveal novel therapeutic targets and novel biomarkers to assist in early disease diagnosis.

This protocol allows for the differentiation of human pluripotent cells into intestinal organoids. The protocol mimics normal human development by differentiating cells into a population of definitive endoderm, hindgut endoderm and then intestinal epithelium. This makes the protocol suitable for studying both intestinal development as well as disease modelling applications.

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of ...

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

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Generation and Characterization of Murine Oral Mucosal Organoid Cultures

Generation and Manipulation of Rat Intestinal Organoids

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications