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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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,

Protocol

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.

  1. Preparation of matrices aliquots and cell culture media
    1. For commercially available animal-derived basement membranes (BMs; Matrix 1-AB, Matrix 2-AB, Matrix 3-AB), prepare aliquots of working volum.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

The authors acknowledge previous training and general recommendations regarding starting hiPSC and organoid work from Drs. Christina Pacak, Silveli Susuki-Hatano, and Russell D'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.

....

Materials

NameCompanyCatalog NumberComments
24-Well Plate (Culture treated, sterile)Falcon353504
37 °C water bathVWR
96-well plate Fisher ScientificFB012931
Advanced DMEM/F12Life Technologies12634
Anti-adherence Rinsing SolutioSTEMCELL Technologies7010
Biological safety cabinet (BSC)Labconco Logic
Brightfield MicroscopeEcho RebelREB-01-E2
BXS0116ATCCACS-1030
Centrifuge with temperature control (4 °C capabilities)ThermoScientific75002441
Conical tubes, 15 mL, sterileThermo Fisher Scientific339650
Conical tubes, 50 mL, sterileThermo Fisher Scientific339652
Cultrex RGF BME, Type 2Bio-techne3533-005-02
Cultrex Stem Cell Qualified RGF BME Bio-techne3434-010-02
D-PBS (Without Ca++ and Mg++)Thermo Fisher Scientific14190144
GeltrexLDEV-Free, hESC-Qualified Reduce Growth FactorGibco A14133-02
GlutaMAX SupplementThermo Fischer Scientific35050-061
Guava Muse Cell Analyzer or another flow cytometry equipment (optional)Luminex0500-3115
HEPES buffer solutionThermo Fischer Scientific15630-056
Heralcell Vios Cell culture incubator (37 °C, 5% CO2)Thermo Scientific 51033775
JMP SoftwareSAS InstituteJMP 16
MATLABMathWorks, IncR2022b
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix LDEV freeCorning 356231
Matrigel Matrix High Concentration (HC), Growth Factor Reduced (GFR) LDEV-freeCorning 354263
mTeSR Plus MediumSTEMCELL Technologies100-0276
Nunclon Delta surface treated 24-well plateThermo Scientific144530
PE Mouse Anti-human CD326 (EpCAM)BD Pharmingen566841
PE Mouse Anti-human CDX2 BD Pharmingen563428
PE Mouse Anti-human FOXA2BD Pharmingen561589
PerCP-Cy 5.5 Mouse Anti-human SSEA4 BD Pharmingen561565
ReLeSRSTEMCELL5872
SCTi003-ASTEMCELL Technologies200-0510
Serological pipettes (10 mL) Fisher Scientific13-678-11E
Serological pipettes (5 mL) Fisher Scientific13-678-11D
STEMdiff Intestinal Organoid Growth MediumSTEMCELL Technologies5145
STEMdiff Intestinal Organoid KitSTEMCELL Technologies5140
Vitrogel Hydrogel MatrixTheWell BioscienceVHM01
VitroGel ORGANOID Discovery KitTheWell BioscienceVHM04-K

References

  1. Hynes, R. O. Integrins: Bidirectional, allosteric signaling machines. Cell. 110 (6), 673-687 (2002).
  2. Frantz, C., Stewart, K. M., Weaver, V. M. The extracellular matrix at a glance. J Cell Sci. 123, 4195-4200 (2010....

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Extracellular MatrixInduced Pluripotent Stem CellsOrganoidsBasement MembraneMatrigelGeltrexCultrexVitroGelXeno freeStem Cell MaintenanceIntestinal Organoid Generation

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