plate preparation for the culture of human induced pluripotent stem cells &#40;hipscs&#41;

Matrix Comparison for Efficient Human Intestinal Organoid Generation from iPSCs

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.

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

인간 유도 만능 줄기 세포(hiPSC) 배양을 위한 플레이트 준비 

먼저 준비된 25ml의 차가운 고급 DMEM/F-12 매체가 들어 있는 튜브를 얼음 위에 놓습니다. 동결해제된 매트릭스를 각 튜브에 추가합니다. 서스펜션을 함께 혼합하여 덩어리가 형성되지 않도록 합니다.이제 희석된 매트릭스 250마이크로리터를 24웰 플레이트의 각 웰에 피펫팅합니다. 코팅 용액이 고르게 펴지도록 플레이트를 기울입니다. 배양 후 혈청학적 피펫을 사용하여 코팅된 표면을 긁지 않고 여분의 용액을 부드럽게 제거합니다.즉시 250 마이크로리터의 따뜻한 완전한 줄기 세포 배지를 각 웰에 피펫팅합니다. 조직 배양 플레이트를 Matrix 4 Xenol-free 하이드로겔로 코팅하려면 먼저 하이드로겔을 실온으로 예열합니다. 한 부피의 3x 줄기 세포 배양 배지가 들어 있는 튜브에 두 부피의 하이드로겔을 추가합니다.용액을 위아래로 피펫팅하여 완전히 혼합합니다. 250 마이크로리터의 하이드로겔을 24웰 조직 배양 플레이트의 웰로 옮깁니다. 그런 다음 플레이트를 기울여 하이드로겔을 고르게 펴 바릅니다.10-15분 후, 코팅된 플레이트는 이제 유도 만능 줄기 세포의 배양을 위해 준비됩니다.

plate-preparation-for-culture-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Bioengineering

Plate Preparation for the Culture of Human Induced Pluripotent Stem Cells &#40;hiPSCs&#41;

44 조회수.  University of Florida. 먼저 준비된 25ml의 차가운 고급 DMEM/F-12 매체가 들어 있는 튜브를 얼음 위에 놓습니다. 동결해제된 매트릭스를 각 튜브에 추가합니다. 서스펜션을 함께 혼합하여 덩어리가 형성되지 않도록 합니다.이제 희석된 매트릭스 250마이크로리터를 24웰 플레이트의 각 웰에 피펫팅합니다. 코팅 용액이 고르게 펴지도록 플레이트를 기울입니다. 배양 후 혈청학적 피펫을 사용하여 코...

비디오: 인간 유도 만능 줄기 세포(hiPSC) 배양을 위한 플레이트 준비 

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.

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) ...