imaging immunostained colorectal cancer&#45;derived organoids in peptide hydrogel for evaluating the effects of self&#45;assembling peptides on cell adhesion

細胞接着および形態学のための生体機能的自己組織化ペプチドの評価

In recent years, self-assembling peptides (SAPs) have emerged as promising biomaterials for tissue engineering applications. SAPs possess unique properties, including spontaneous formation of nanofibers, biocompatibility, biodegradability, and non-immunogenicity, making them attractive candidates for scaffold development1. SAPs have been previously used together with various types of cells, and notably, reported ultrashort SAPs have facilitated the encapsulation of stem cells while upholding their pluripotency over prolonged periods encompassing more than 30 passages and with minimal incidence of chromosomal aberrations2,3,4. Hence, adapting SAPs for their use in organoid culture constituted a rational subsequent step.
Organoids are complex three-dimensional structures that arise from single pluripotent cells. These structures give rise to various cell types, which then self-organize to replicate the developmental processes of embryonic and tissue growth in vitro5. This innovative framework has evolved into a formidable tool for in vitro investigations, efficiently conserving the genetic, phenotypic, and behavioral attributes characteristic of in vivo organs6. Nevertheless, a principal impediment in the bench-to-bedside transition of organoid-based findings is the need for reproducibility7. This variation in results is primarily ascribed to the difficulty in fully differentiating human pluripotent stem cells into specialized cell lineages, compounded by the absence of authentic tissue architecture and the inherent complexity encountered in organoid models. Given the rise in popularity of stem-cell- and organoid-based studies8, there has been an escalated demand for biomaterials with dynamic mechanical properties and integrin-like adhesion sites or scaffolds with controlled degradability7,9. These attributes can be effectively tuned through the strategic utilization of biofunctionalized SAPs.
SAPs are short sequences of amino acids with the inherent ability to spontaneously organize into well-defined structures10. These peptides often contain alternating hydrophobic and hydrophilic residues, which drive their self-assembly through non-covalent interactions, including hydrogen bonding, electrostatic interactions, and hydrophobic effects11,12. The self-assembly process of these peptides is primarily driven by the need to minimize the system&#39;s free energy. When in an aqueous environment, the hydrophobic residues tend to cluster together to minimize their exposure to water, while the hydrophilic residues interact with the surrounding water molecules. This phenomenon leads to the formation of various nanostructures. In this case, ultrashort self-assembling peptides form nanofibers with characteristics that depend on the peptide sequence and environmental conditions2,12,13,14,15,16. These peptides adopt a &#946;-turn conformation, where individual peptide strands align parallel or antiparallel to each other, stabilized by hydrogen bonds (Figure 1). The presence of positive ions accelerates the self-assembling processes that lead to nanofiber formation.
Peptide nanofibers have been identified as possessing an innate ability to entrap significant volumes of water. This characteristic facilitates the generation of a hydrogel, which subsequently manifests as a biocompatible and biodegradable three-dimensional space conducive to cellular proliferation and growth. These self-assembling peptide nanofibers intricately emulate the topographical features inherent to the natural extracellular matrix (ECM) environment1. This resemblance endows cells with an environment that imitates their physiological habitat, further promoting optimal cellular activities17. Furthermore, the versatility inherent to these peptides permits a considerable degree of tunability4. Such adaptability enables changes to the matrix&#39;s properties, such as stiffness, gelation kinetics, and porosity. These adaptations are achieved through modifications to the peptide sequence. Consequently, self-assembling peptides (SAPs) have emerged as pivotal components in contemporary biomaterial science, extensively used as scaffolds for tissue regeneration and cellular culture13,17.
One of the critical advantages of self-assembling peptides is their ability to be easily synthesized and modified at the molecular level. This advantage allows for the incorporation of specific functional groups or bioactive motifs into the peptide sequence, enabling the design of peptides with tailored properties and functionalities18,19,20 (Figure 1B). For example, biofunctionalized SAPs can be designed to mimic the ECM and promote differentiation using the RGD peptide19,21. Peptide Ben-IKVAV has also been reported to significantly increase the expression of neuronal-specific markers due to its ligand-specific motiety22. These peptides can also be engineered to display bioactive molecules, such as integrin-binding peptides, to enhance cell survival23. Finally, other biofunctionalized SAPs have been developed to promote angiogenesis by including the IKVAV and YIGSR motifs in their structures24.
Biofunctionalized SAPs reported in an earlier publication show that self-assembling can be further modified by including the na&#239;ve peptide from which the biofunctionalized SAP was derived25. These SAP mixtures can also provide a wide array of SAP formulations that vary in their biochemical activity and physicochemical properties. For instance, the amphiphilic peptide IIFK is an ultrashort SAP that can withstand biofunctionalization with various motifs, exemplified by the incorporation of the IKVAV motif (Figure 1B). Both peptides can form nanofibers, both alone and combined. Each formulation results in hydrogels with varying physical properties (Figure 2)
However, because of the extensive array of potential permutations and alternatives obtained from the biofunctionalization of SAPs, there is a need to provide a fast and cost-effective option for organoid testing. One candidate for such intensive and cost-effective organoid evaluation is the SW1222 cell line, derived from human colorectal adenocarcinoma26,27. SW1222 cells possess characteristics that enable their aggregation into 3D structures resembling organoids, making them an ideal model for studying tissue development and regenerative medicine applications. SW1222 cells have been identified as capable of generating organoids owing to their intrinsic overexpression of the LGR5 gene27. The creation of organoids from individual LGR5+ stem cells has been convincingly exhibited before, as well as the propensity of SW1222 cells to achieve morphological characteristics of a colorectal cancer organoid28.
In this methods paper, we present detailed protocols for evaluating and characterizing the effects of various biofunctional peptides on cell adhesion and lumen formation using SW1222 cells (Figure 3). By providing step-by-step procedures and imaging analysis methods, we hope to offer valuable insights into the biofabrication of organoids and the evaluation of simplistic SAP matrices for organoid culture.

著者スポットライト:結腸直腸癌オルガノイドのための超短ペプチドマトリックスの最適化

超短自己組織化ペプチドマトリックス中のSW1222細胞株由来結腸直腸癌オルガノイドの作製とキャラクタリゼーション

Our research aims to evaluate ultrashort self-assembling peptides as matrices for Colorectal Cancer Organoid Cultures. We address questions about organoid morphology, for viability, proliferation, and the adhesion and the impact of biofunctionalization. The goal is to optimize peptide matrix compositions for effective organoid growth, and to contribute to regenerative medicine.Cutting-edge technologies in our field involve 3D bioprinting for precise organic fabrication, microfluidic platforms for dynamic culture environments, and advanced imaging tools like atomic force microscopy and multiphoton microscopy. CRISPR-Cas9 gene editing refines genetic modification, while single cell RNA sequencing provides in-depth molecular insights. Current experimental challenges include enhancing organoid reproducability, refining vascularization for larger constructs, and mimicking complex tissue architecture.Overcoming these limitations in long-term culture and incorporating immune components into models are active areas. Achieving standardized protocols and addressing ethical concerns opposing ongoing challenges in this field. Significant findings in our field include demonstrating the suitability of ultrashort self-assembling peptides for organoid cultures.We have shown the versatility, reproducability, and stability of these peptides, providing a platform for fine tuning micro-environments. This contributes to advancing organoid-based studies, regenerative medicine, and biofunctionalized hydrogel research. Our protocol bridges and research gap, whereby providing a systematic approach to evaluating organoids in ultrashort self-assembling peptide matrices.It addresses the need for standardized methods in assessing cell behavior within these matrices, aiding in durational design of peptide-based hydrogels for optimal organoid growth and advancing regenerative medicine studies.

細胞接着に対する自己組織化ペプチドの影響を評価するためのペプチドハイドロゲル中の免疫染色された結腸直腸癌由来オルガノイドのイメージング

まず、染色したSW1222由来オルガノイドサンプルを落射蛍光顕微鏡で10倍でイメージングします。Rhod チャネルと DAPI チャネルのみを使用します。ImageJソフトウェアを起動し、プラグインをクリックしてからマクロをクリックします。次に、ImageJ ROI コンバーターを実行するために実行します。py ファイルを Cell Pose GitHub サイトからダウンロードできます。ウィンドウが表示されたら、コロンオルガノイドフォルダから最初のファイルを選択し、[開く]をクリックします。次に、セグメンテーションを選択します。npy ファイルを開き、[開く] をクリックします。次に、ROIマネージャーウィンドウでselect allを押し、measureを押します。次に、[ファイル]をクリックし、次に結果ウィンドウで[名前を付けて保存]をクリックして結果を保存します。コロンオルガノイド内腔のフォルダ内の同じ画像に対して画像変換を繰り返します。次に、Origin Proソフトウェアをロードし、データをクリックしてから、ファイルのインポートをクリックしてインポートウィザードを見つけます。同じ画像のオルガノイドとルーメン形状のプロパティに対応する両方のCSVファイルを選択します。ルーメン形状のプロパティをコロニー結果列にコピーして、各ルーメン測定値を対応するコロニーとペアにします。新しい列で、ルーメンとコロニーの領域を分割して、対応するコロニーの相対的なルーメン領域を求めます。各コロニーの真円度とルーメン面積に対応するカラムを選択します。次に、プロット、基本 2D、および散布図を順番にクリックします。プロットウィンドウを右クリックし、プロットの詳細を選択します。centroid proタブをクリックし、サブセットの重心点の表示、データポイントへの接続、および楕円の表示を読み取るオプションを選択します。2Dで培養した細胞は、コロニーの真円度に非常に高いばらつきが見られました。マトリゲルで培養した細胞は、内腔の相対的なサイズに対してより広範な分布を示していました。

imaging-immunostained-colorectal-cancerderived-organoids-peptide

Research

JoVE Journal

Bioengineering

Imaging Immunostained Colorectal Cancer&#45;Derived Organoids in Peptide Hydrogel for Evaluating the Effects of Self&#45;Assembling Peptides on Cell Adhesion

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...

生物工学

SW1222 Cell Line&#45;Derived Colorectal Cancer Organoid Fabrication in Ultrashort Self-Assembling Peptide Matrix

SW1222 超短自己組織化ペプチドマトリックスにおける細胞株由来結腸直腸癌オルガノイド作製

Immunostaining of Colorectal Cancer&#45;Derived Organoids in Peptide Hydrogel

ペプチドハイドロゲル中の大腸がん由来オルガノイドの免疫染色