3d cell culture methods and protocols methods in molecular biology 2764 2nd edition zuzana sumbalov
Zuzana Sumbalova Koledova
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Epithelial Cell Culture Methods and Protocols Methods in Molecular Biology 2749 Mario Baratta
School of Life and Medical Sciences, University of Hertfordshire, Hatfield, Hertfordshire, UK
For further volumes: http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-bystep protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible stepby-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.
Editor Zuzana Sumbalova Koledova
3D Cell Culture
Methods and Protocols
2nd ed. 2024
Editor
Zuzana Sumbalova Koledova
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Laboratory of Tissue Morphogenesis and Cancer Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic
ISSN 1064-3745
e-ISSN 1940-6029
Methods in Molecular Biology ISBN 978-1-0716-3673-2 e-ISBN 978-1-0716-3674-9
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained
herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface
Welcome to the new edition of 3D Cell Culture. Since the publication of the last edition, the field has witnessed remarkable progress, with groundbreaking discoveries, novel methodologies, and transformative applications. In particular, the use of organoid cultures has become more widespread across laboratories, organoid models have been developed for many more organs, new hydrogels and devices for 3D culture have been invented, and the organoid systems have been improved by incorporating more components of the tissue microenvironment into the in vitro culture. All in all, there was a lot of new material for the book.
However, when I was invited to serve as the editor for this edition of the 3D Cell Culture book, I hesitated. One of my concerns was the amount of time and work this endeavor would require. I could well remember that in 2017, after completing the first edition of the book, I had promised myself that I would never again take on this enormous task; however, the memory of the book labor pain has faded over the years and the good memories of networking and communicating with the contributors have prevailed. In addition, I felt that I had gained enough experience with the first edition of the book, as well as with my other editorial work for scientific journals, to be able to take on this challenge more easily.
My second hesitation was the timing of the offer. It was shortly after the world had begun to recover from the COVID-19 pandemic. The days of mandatory face masks, reduced interpersonal contact, and research labs closed or operating at a bare minimum were over. After almost 2 years of spending too much time at the computer writing publications (the researchers’ lemonade made from the lemons of the pandemic), the scientists were finally allowed to return to their lab benches full time. Would they be willing to sit at their desks again to write the book chapters? Fortunately, they were! Many of the authors of the first edition expressed their interest in contributing to the second edition. Re-establishing face-toface international meetings allowed me to meet and recruit new contributors. And social media helped to spread the word about the call for papers and attract several more authors to this project.
As the editor, I am immensely grateful for the dedication and expertise of all the contributors who have made this edition possible. Their willingness to share detailed protocols with intricate details and tips will
help other researchers to successfully apply these techniques in their laboratories, paving the way for the expansion of our scientific knowledge and for revolutionary breakthroughs with the potential to reshape the future of medicine.
In this edition, we delve deeper into specific methods and techniques for 3D cell culture, with applications spanning multiple disciplines: from basic science through cancer research and drug development to tissue engineering and regenerative medicine. The dynamic nature of the field and the demand for tools to address specific scientific questions have led to the emergence of new challenges, prompting researchers to innovate and collaborate across traditional boundaries. This edition captures the essence of this effort, offering insights into cutting-edge technologies and their transformative potential. Experts in the field contribute their hands-on experience in topics such as biofabrication, organoids, microfluidic systems, bioprinting, and image analysis. With each chapter, readers will learn specific techniques that they can easily apply in their laboratories.
I would like to express my appreciation to the readers whose curiosity and quest for knowledge drive the continued development of this field. It is my sincere hope that this edition will serve as a useful resource and source of inspiration for experienced researchers, newcomers to the field, and anyone intrigued by the convergence of biology, engineering, and medicine. May this issue stimulate new ideas, foster interdisciplinary collaborations, and ultimately contribute to the improvement of human health and wellbeing.
Zuzana Sumbalova Koledova
Contents
1 3D Cell Culture: Techniques For and Beyond Organoid Applications
Zuzana Sumbalova Koledova
Part I Hydrogels and Scaffolds for 3D Cell Culture
2 Preparation of Corneal Tissue Matrix Bioink for 3D Bioprinting
Shibu Chameettachal and Falguni Pati
3 Decellularization of Pig Lung to Yield Three-Dimensional Scaffold for Lung Tissue Engineering
Katarína Čimborová, Hana Kotasová, Vendula Pelková, Veronika
Sedláková and Aleš Hampl
4 Three-Dimensional Hanging Drop Spheroid Plates for Easy Chimeric Antigen Receptor (CAR) T Cytotoxicity Assay
Zhenzhong Chen and Sungsu Park
5 Static 3D Osteoblast Cell Culture on 3D Printed Titanium Scaffolds
Carolina Oliver-Urrutia, Anna Diez-Escudero, Zuzana Sumbalova
Koledova, Ladislav Čelko, Nils P. Hailer and Edgar B. Montufar
6 Modeling Acute Myeloid Leukemia Using StarPEG-Heparin Hydrogels
P. Lewen Holloway, Akhilandeshwari Ravichandran, Julien Clegg, Claudia Bruedigam and Laura J. Bray
Part II 3D Organoid and Organotypic Cultures
7 A Decision Tree to Guide Human and Mouse Mammary Organoid Model Selection
Marika Caruso, Kamyab Saberiseyedabad, Larissa Mourao and Colinda
L. G. J. Scheele
8 Fibroblast-Epithelium Co-culture Methods Using Epithelial Organoids and Cell Line–Derived Spheroids
Jakub Sumbal and Zuzana Sumbalova Koledova
9 Differentiation of Fibroblasts to Adipocytes in 3D for a Co-culture with Mammary Organoids and Immunohistological Analysis
Matea Brezak, Lukas Kubec and Zuzana Sumbalova Koledova
10
Organotypic 3D Cell Culture of the Embryonic Lacrimal Gland
Alison Kuony, Matea Brezak, René-Marc Mège and Zuzana Sumbalova
Koledova
11 Renal Organoids from Whole Kidney Cells
Liang Chen
12 Organotypic 3D Ex Vivo Co-culture Model of the Macro-metastasis/ Organ Parenchyma Interface
Raquel Blazquez, Daniela Sparrer, Jessica Sonbol, Jürgen Philipp, Florian Schmieder and Tobias Pukrop
Part III Functional Experiments in 3D Cell Cultures
13 Addressing the Mechanical Interaction Between Cancer-Associated Fibroblasts and Cancer Cells by Laser Ablation
Carlos Perez-Gonzalez and Jorge Barbazán
14 Double-Barrel Perfusion System for Modification of Luminal Contents of Intestinal Organoids
Nicholas J. Ginga and Raleigh Slyman
15 Mechanical Actuation of Organoids in Synthetic Microenvironments
Francesca Sgualdino, Lorenzo Mattolini, Brian Daza Jimenez, Kieran
Patrick, Abdel Rahman Abdel Fattah and Adrian Ranga
Part IV Microfluidic and Bioprinting Approaches for 3D Cell Culture
16 3D Breast Cancer Model on Silk Fibroin–Integrated Microfluidic Chips
Eylul Gulsen Yilmaz and Fatih Inci
17 Tumor-Microenvironment-on-Chip Platform for Assessing Drug Response in 3D Dynamic Culture
Hakan Berk Aydin, Hye-ran Moon, Bumsoo Han, Altug Ozcelikkale and Ahmet Acar
18 Embedded 3D Bioprinting for Engineering Miniaturized In Vitro Tumor Models
Maria V. Monteiro, Marta Rocha, Vítor M. Gaspar and João F. Mano
Part V Imaging and Image Analysis of 3D Cell Cultures
19 Real-Time Cell Cycle Imaging in a 3D Cell Culture Model of Melanoma, Quantitative Analysis, Optical Clearing, and Mathematical Modeling
Loredana Spoerri, Kimberley A. Beaumont, Andrea Anfosso, Ryan J. Murphy, Alexander P. Browning, Gency Gunasingh and Nikolas K. Haass
20 Quantitative Analysis of Whole-Mount Fluorescence-Stained Tumor Spheroids in Phenotypic Drug Screens
Elina Nuernberg, Roman Bruch, Mathias Hafner, Ruediger Rudolf and Mario Vitacolonna Index
Contributors
Abdel Rahman Abdel Fattah
Laboratory of Bioengineering and Morphogenesis, Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven, Belgium
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
Ahmet Acar
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey
Andrea Anfosso
The Centenary Institute, Sydney, NSW, Australia
Hakan Berk Aydin
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey
Jorge Barbazán
Translational Medical Oncology Group (ONCOMET), Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain
Kimberley A. Beaumont
The Centenary Institute, Sydney, NSW, Australia
Uniquest, The University of Queensland, Brisbane, QLD, Australia
Raquel Blazquez
Department of Internal Medicine III, Hematology and Medical Oncology, University Hospital Regensburg, Regensburg, Germany
Laura J. Bray
School of Mechanical, Medical and Process Engineering, Queensland University of Technology, Kelvin Grove, QLD, Australia
ARC Training Centre for Cell and Tissue Engineering Technologies, Queensland University of Technology, Kelvin Grove, QLD, Australia
Matea Brezak
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Alexander P. Browning
Mathematical Sciences, Queensland University of Technology, Brisbane, QLD, Australia
Roman Bruch
Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany
Institute for Automation and Applied Informatics, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany
Claudia Bruedigam
Cancer Program, Queensland Institute of Medical Research (QIMR)
Berghofer Medical Research Institute, Herston, QLD, Australia
School of Medicine, University of Queensland, Herston, QLD, Australia
Marika Caruso
VIB-KU Leuven Center for Cancer Biology, Department of Oncology, Leuven, Belgium
Ladislav Čelko
Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
Shibu Chameettachal
Department of Biomedical Engineering, Indian Institute Technology Hyderabad, Sangareddy, Telangana, India
Liang Chen
Urology Department, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Zhenzhong Chen
School of Mechanical Engineering, Sungkyunkwan University, Suwon, Korea
Katarína Čimborová
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Julien Clegg
School of Mechanical, Medical and Process Engineering, Queensland
University of Technology, Kelvin Grove, QLD, Australia
ARC Training Centre for Cell and Tissue Engineering Technologies, Queensland University of Technology, Kelvin Grove, QLD, Australia
Anna Diez-Escudero
Department of Surgical Sciences – Orthopaedics, Uppsala University Hospital, Uppsala, Sweden
Vítor M. Gaspar
Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
Nicholas J. Ginga
Department of Mechanical & Aerospace Engineering, The University of Alabama in Huntsville, Huntsville, AL, USA
Gency Gunasingh
Frazer Institute, The University of Queensland, Brisbane, QLD, Australia
Nikolas K. Haass
Frazer Institute, The University of Queensland, Brisbane, QLD, Australia
The Centenary Institute, Sydney, NSW, Australia
Mathias Hafner
Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany
Nils P. Hailer
Department of Surgical Sciences – Orthopaedics, Uppsala University Hospital, Uppsala, Sweden
Aleš Hampl
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
International Clinical Research Center, St. Anne’s University Hospital, Brno, Czech Republic
Bumsoo Han
School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA
Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA
P. Lewen Holloway
School of Mechanical, Medical and Process Engineering, Queensland University of Technology, Kelvin Grove, QLD, Australia
Fatih Inci
UNAM-National Nanotechnology Research Center, Bilkent University, Ankara, Turkey
Institute of Materials Science and Nanotechnology, Bilkent University, Ankara, Turkey
Brian Daza Jimenez
Laboratory of Bioengineering and Morphogenesis, Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven, Belgium
Hana Kotasová
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Lukas Kubec
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Alison Kuony
Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France
João F. Mano
Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
Lorenzo Mattolini
Laboratory of Bioengineering and Morphogenesis, Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven, Belgium
René-Marc Mège
Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France
Maria V. Monteiro
Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
Edgar B. Montufar
Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
Hye-ran Moon
School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA
Larissa Mourao
VIB-KU Leuven Center for Cancer Biology, Department of Oncology, Leuven, Belgium
Ryan J. Murphy
Mathematical Sciences, Queensland University of Technology, Brisbane, QLD, Australia
Elina Nuernberg
Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany
Center for Mass Spectrometry and Optical Spectroscopy (CeMOS), Mannheim University of Applied Sciences, Mannheim, Germany
Carolina Oliver-Urrutia
Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
Altug Ozcelikkale
Department of Mechanical Engineering, Middle East Technical University, Ankara, Turkey
Graduate Program of Biomedical Engineering, Middle East Technical University, Ankara, Turkey
Sungsu Park
School of Mechanical Engineering, Sungkyunkwan University, Suwon, Korea
Falguni Pati
Department of Biomedical Engineering, Indian Institute Technology Hyderabad, Sangareddy, Telangana, India
Kieran Patrick
Laboratory of Bioengineering and Morphogenesis, Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven, Belgium
Vendula Pelková
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Carlos Perez-Gonzalez
Cell Biology and Cancer Unit, Institut Curie, PSL Research University, CNRS, Paris, France
Jürgen Philipp
Fraunhofer Institute of Materials and Beam Technology IWS, Dresden, Germany
Tobias Pukrop
Department of Internal Medicine III, Hematology and Medical Oncology, University Hospital Regensburg, Regensburg, Germany
Division of Personalized Tumor Therapy, Fraunhofer Institute for Toxicology and Experimental Medicine, Regensburg, Germany
Adrian Ranga
Laboratory of Bioengineering and Morphogenesis, Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven, Belgium
Akhilandeshwari Ravichandran
School of Mechanical, Medical and Process Engineering, Queensland University of Technology, Kelvin Grove, QLD, Australia
Marta Rocha
Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
Ruediger Rudolf
Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany
Center for Mass Spectrometry and Optical Spectroscopy (CeMOS), Mannheim University of Applied Sciences, Mannheim, Germany
Kamyab Saberiseyedabad
VIB-KU Leuven Center for Cancer Biology, Department of Oncology, Leuven, Belgium
Colinda L. G. J. Scheele
VIB-KU Leuven Center for Cancer Biology, Department of Oncology, Leuven, Belgium
Florian Schmieder
Fraunhofer Institute of Materials and Beam Technology IWS, Dresden, Germany
Veronika Sedláková
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Francesca Sgualdino
Laboratory of Bioengineering and Morphogenesis, Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven, Belgium
Raleigh Slyman
Department of Mechanical Engineering, Stanford University, Stanford, CA, USA
Jessica Sonbol
Department of Internal Medicine III, Hematology and Medical Oncology, University Hospital Regensburg, Regensburg, Germany
Daniela Sparrer
Department of Internal Medicine III, Hematology and Medical Oncology, University Hospital Regensburg, Regensburg, Germany
Loredana Spoerri
Frazer Institute, The University of Queensland, Brisbane, QLD, Australia
Jakub Sumbal
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Laboratory of Genetics and Developmental Biology, Institut Curie, INSERM U934, CNRS UMR3215, Paris, France
Sorbonne Université, Collège Doctoral, Paris, France
Zuzana Sumbalova Koledova
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Laboratory of Tissue Morphogenesis and Cancer, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic
Mario Vitacolonna
Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany
Center for Mass Spectrometry and Optical Spectroscopy (CeMOS), Mannheim University of Applied Sciences, Mannheim, Germany
Eylul Gulsen Yilmaz
UNAM-National Nanotechnology Research Center, Bilkent University, Ankara, Turkey
Institute of Materials Science and Nanotechnology, Bilkent University, Ankara, Turkey
In the rapidly evolving landscape of cell biology and biomedical research, three-dimensional (3D) cell culture has contributed not only to the diversification of experimental tools available but also to their improvement toward greater physiological relevance. 3D cell culture has emerged as a revolutionary technique that bridges the long-standing gap between traditional two-dimensional (2D) cell culture and the complex microenvironments found in living organisms. By providing conditions for establishing critical features of in vivo environment, such as cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper tissue-like architecture and differentiated function of cells. Since the early days of 3D cell culture in the 1970s, the field has witnessed remarkable progress, with groundbreaking discoveries, novel methodologies, and transformative applications. One particular 3D cell culture technique has caught the attention of many scientists and has experienced an unprecedented boom and enthusiastic application in both basic and translational research over the past decade – the organoid technology. This book chapter provides an introduction to the fundamental concepts of 3D cell culture including organoids, an overview of 3D cell culture techniques, and an overview of methodological- and protocol-oriented chapters in the book 3D Cell Culture.
1 Introduction
In the field of cell and cancer biology, traditional 2D cell culture techniques have long been the standard for studying cellular behavior and responses to various stimuli. However, these flat and artificial environments often fail to accurately mimic the complex 3D architecture and microenvironment that cells encounter in living organisms. This limitation has led to a growing interest in 3D cell culture systems, which overcome the gap between conventional 2D cultures and the intricate in vivo conditions, and to a 3D culture and organoid revolution in basic and translational research.
The tissue microenvironment, encompassing elements such as the extracellular matrix (ECM) and neighboring cells, plays a crucial role in shaping tissue structure and function, and it is pivotal in regulating tissue growth and development [1]. However, traditional 2D cell cultures, while straightforward to set up, lack the necessary environmental context and structural organization, resulting in alterations in cell behavior For example, when cultivated as flat monolayers in 2D, normal epithelial cells often lose their distinct characteristics and become highly adaptable and resembling tumor cells [2, 3]. Additionally, malignant cells in 2D culture exhibit differences from their solid tumor counterparts [4].
To address these limitations and to provide a more physiologically relevant alternative to 2D cell culture, three-dimensional (3D) cell culture techniques have been developed. By taking into account the critical interactions between cells and their surroundings, 3D cultures allow cells to mimic several essential features
found in tissues. These include aspects such as shape, differentiation, polarity, rate of proliferation, gene expression, genomic profiles, as well as the cellular diversity and the presence of nutrient and oxygen gradients found in tumors [5].
Critically, 3D cell culture bridges the gap between 2D cell culture and animal models. 3D cell cultures incorporate biologically relevant interactions while still allowing for easy genetic manipulation, biochemical analysis, and imaging. By creating a controlled microenvironment that mimics in vivo conditions, 3D cell cultures have emerged as a valid alternative to animal testing. Furthermore, the ability to engineer specific microenvironments in 3D enables researchers to explore questions that are challenging to address within living organisms [5].
Among various discoveries, 3D cell culture models have illustrated the fundamental importance of the cellular and ECM microenvironment in influencing cell behavior. For instance, when tumor cells were embedded as single cells in 3D collagen, they exhibited individual migration and invasion. When embedded as small clusters, they invaded collectively. And when embedded as large aggregates, some cells invaded, while others underwent necrosis [6]. Similarly, mouse mammary epithelial cells displayed in vivo-like structural organization and functional differentiation, including milk secretion into lumens, only when cultured in a reconstituted basement membrane matrix, as opposed to collagen I gels or monolayers [7]. Subsequent studies have consistently demonstrated significant effects of ECM composition and stiffness on cell signaling and behavior [8–12].
2 The History and Development of 3D Cell Cultures
The main prerequisite for the development of 3D cell cultures was the desire of researchers to recapitulate organogenesis in vitro and their experiments beginning twentieth century, which led to the understanding of the importance of the cellular microenvironment in the regulation of tissue-specific morphogenetic programs [13]. Early attempts at in vitro tissue culture included organ/tissue fragment culture using the hanging drop method, watch glass culture, and lens paper culture on metal grids [14–16]. In the mid-twentieth century, researchers observed that cells obtained from dissociated organs and cultured in suspension were capable of reaggregating and forming the structural pattern of the original tissue [17].
In 1956, Ehrman and Gey, who developed a method for reconstituting of collagen from rat tails, demonstrated superior growth and survival of cells grown on collagen gels [18]. However, several cell types were losing their differentiation functions after a few days of culture on thick, rigid collagens [19]. Only when the gels were detached and floated in the medium, the epithelial cells (hepatocytes) retained their differentiated status [20]. When a similar experiment was performed with mammary epithelial cells isolated from pregnant mice (and with addition of lactogenic hormones to the medium), it was found that the mammary cells were able to express milk protein in floating collagen gels, whereas milk protein expression was not achieved in cells cultured on attached collagen gels or plastic dishes [21]. These experiments suggested that mechanical properties of the ECM gels influenced cell fate. In 1977, a new ECM gel with basement membrane properties was isolated – a laminin-rich reconstituted basement membrane matrix now known as Matrigel [22]. When mammary epithelial cells were cultured on it, they efficiently expressed milk protein as well as formed 3D mammary gland-like structures [7], giving evidence that the ECM controls gene expression as well as morphogenesis. Moreover, with these 3D cultures of primary mammary epithelial cells in ECM gels, the first organoid cultures were born.
Since their first use in the 1970s in the form of floating collagen gels, 3D cell culture systems have undergone significant development and continue to evolve, with the ultimate goal of recreating entire organs in culture. Several scientific fields, such as bioengineered materials, nanotechnology, microfluidics, and 3D bioprinting, have contributed tools and technologies to manipulate and control the cellular microenvironment (including its chemistry, geometry, and mechanics) at multiple levels and scales with greater precision. These advances have led to the creation of numerous 3D cell culture systems tailored to specific scientific requirements.
3 Materials and
Methods
for 3D Cell Culture
Several distinct types of 3D cell culture methods can be recognized according to the variations in cellular input, cell culture formats, and the characteristics of the 3D microenvironment. The most straightforward and historically oldest form of 3D cell culture involves ex vivo or in vitro cultivation of entire organs, tissue explants, or slices of tissue, often with mechanical support. Scientists have been culturing whole organs since the late 1920s to investigate organ development mechanisms [15]. However, while this approach permits experimental observation and manipulation within native tissues, tissue viability is limited, and interpreting results from these complex tissues can be challenging. To adopt a more reductionist approach, organs, tissues, or tumors can be deconstructed into their individual cellular subpopulations and the ECM These components can then be selectively recombined in vitro to address specific research questions.
For the cellular component of 3D cell cultures, both primary cells and established cell lines can be used. Primary cells, either in the form of primary organoids (tissue fragments) or individual primary cells, are obtained by mechanical disruption and enzymatic digestion of donor tissue. Enzymatic digestion employs enzymes, such as collagenase, dispase, trypsin, hyaluronidase, or elastase, to disrupt the ECM and release cells embedded in it. Mechanical dissociation often precedes (e.g., cutting tissue with scalpels) or complements (e.g., repetitive pipetting) the enzymatic dissociation process and helps to increase the reaction surface for enzymatic action.
Dissociated primary cells, including tumor cells, as well as established cell lines, can be cultured either randomly dispersed within ECM, grown as sheets, or aggregated into spheroids (clusters of cells) using various methods. The most common techniques for spheroid generation prevent cell-to-dish surface adhesion and promote cell-to-cell attachment by culturing cells on non-adherent surfaces, such as agarosecoated flat-bottom plates, poly(2-hydroxyethyl methacrylate)-coated plates, ultra-low adhesion flat-, U-, or V-bottomed plates, micropatterned plates, and hanging drops. Alternatively, spheroids can be formed through spontaneous aggregation or by employing spinner flasks and rotary cell culture systems [23].
It is important to distinguish between different types of 3D cellular structures and do not use the terms such as organoid and spheroid interchangeably Organoids are self-organizing 3D cellular structures composed of two or more types of cells that resemble miniaturized organ units in architecture and function. Organoids can be established from primary tissue fragments (explants), multipotent tissue stem cells, or pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells. Spheroids, on the other hand, are spherical masses of cells formed by the proliferation or aggregation of usually a single cell type. Spheroids lack tissue-like architecture and self-organization and exhibit limited tissue functionality [24]. When organoids or spheroids are combined with more cell types into 3D cellular structures, aggregates or assembloids are formed. Aggregates are any 3D cellular structures formed by aggregation of organoids or spheroids with cells of another cell type [25]. Assembloids are more complex self-organizing 3D cellular systems that result from the integration of multiple types of organoids or the combination of organoids with missing cell types or primary tissue explants, including tumors [26, 27].
Regarding 3D cell culture itself, cellular components can be cultured in several ways: (1) suspended on non-adherent surfaces or in bioreactors, (2) embedded within hydrogels, (3) placed on mechanical supports, such as tissue culture inserts for air-liquid interface culture, or (4) situated on scaffolds and microcarriers. Hydrogels used for 3D cell culture include natural ECM, such as collagen, reconstituted basement membrane matrix (Matrigel) and decellularized ECM, alginate, biohybrid and synthetic hydrogels. Matrigel, composed of laminin, collagen IV, entactin, perlecan and growth factors [28], has been the most widely used hydrogel in organoid culture. However, its animal-derived origin makes it inherently heterogeneous, poorly defined, and variable between batches [29], which affects reproducibility of the experimental results. Therefore, alternatives to Matrigel have been explored and developed, including combinations of recombinant ECM proteins or synthetic hydrogels with defined composition and tunable stiffness [29].
The mechanical properties of hydrogels have major influence on cell behavior and fate in 3D cell culture [30, 31] and can be controlled in some hydrogel types, for example, in collagen-containing gels by adjusting collagen concentration, or calcium concentration in alginate gels, or by detaching an attached gel to create a floating-gel culture. Furthermore, various techniques, such as photomasks, micro-needles, two-photon polymerization, or soft lithography, can be employed to micropattern the gel, creating specific patterns such as alternating low-stiffness and high-stiffness regions or areas that permit or restrict cell growth [5].
Technological advances have introduced microfluidic devices and 3D bioprinting to the spectrum of 3D cell culture methods. Microfluidic devices are utilized for high-throughput spheroid production, embedding
in ECM, experimental testing, or the creation of biomimetic models such as organs-on-chip. Microfluidic organs-on-chip consist of continuously perfused microchannels lined with living cells arranged to mimic minimal functional units, simulating tissue- and organ-level functions [32]. These devices replicate the multicellular architectures, tissue interfaces, physiochemical microenvironments, and vascular perfusion found in the human body, enabling the study of organ-level responses while facilitating precise manipulation of the cell culture environment and automation. Microfluidic devices are fabricated using a variety of techniques. These include soft lithography, injection molding, micromolding, microetching, laser etching, micromilling, photopolymerization, and others [33]. 3D bioprinting is an additive manufacturing technique used to precisely position biological materials, biochemicals, and living cells layer by layer to create 3D biomimetic structures [34]. The primary strategies for 3D bioprinting include inkjet, extrusion, laserassisted, and stereolithography bioprinting [34, 35].
4 Expertise Covered in This Book
This book provides detailed protocols for various 3D culture techniques, including scaffold-free and scaffold-based approaches (and methods for fabricating such scaffolds), organoids, microfluidic systems, and bioprinting, as well as methods for analyzing the results (Table 1).
Table 1 Overview of 3D cell culture methods presented in this book
Sirius red, Alizarin red and IF staining LDH and ALP assays
Fluorescence staining Viability assays
method(s)
or functional assays ECM/hydrogel Format Fluid dynamic
aggregation
8 rBM (Matrigel) Embedded Static 3D co-culture Nonadherent plate (polyHEMAcoated)
Mouse mammary organoids (normal and tumor) and mammary fibroblasts
9 rBM (Matrigel) Embedded Static 3D culture and 3D co-culture No No
10 rBM (Matrigel) Embedded Static 3D co-culture No
techniques
Primary mouse mammary organoids (normal and tumor), primary mammary fibroblasts, breast cancer line, e.g., MCF7-ras No No
Primary mouse mammary fibroblasts and organoids Fluorescence and Oil Red O staining No
Mouse embryonic lacrimal organoids and mesenchymal cells
Primary mouse embryonic lacrimal organoids and mesenchymal cells No No
11 Decellularized ECM Embedded Static ECM decellularization and 3D culture Hanging drop plate Human kidney cells Human kidney cells No No
12 rBM (Matrigel) Embedded, mechanically supported Static Organotypic tissue slice coculture No No
Mouse brain slices and tumor cells IF staining No 13 PAA gels coated with collagen I On top Static 3D co-culture No Cancer associated fibroblasts and tumor cells
Cancer associated fibroblasts and tumor cells No Laser ablation 14 rBM (Matrigel) Embedded Dynamic Organoid perfusion device fabrication No No Oganoids, e.g., intestinal No Organoid perfusion 15 PEG hydrogel Embedded on stretchable membranes Static Mechanical actuation of organoids No
hiPSCs IF staining Bead tracking experiments
Silk fibroin Microfuidic Dynamic Fabrication of microfluidic device
No Viability assay 19 Collagen Embedded Static 3D real-time imaging by confocal microscopy Agarosecoated plate No Melanoma cell line No No
ALP alkaline phosphatase, ECM extracellular matrix, hiPSCs human induced pluripotent stem cells, GelMA gelatin methacrylate, IF immunofluorescence, LDH lactate dehydrogenase, PAA polyacrylamide, PEG polyethylene glycol, rBM reconstituted basement membrane
The first part of the book on Hydrogels and Scaffolds for 3D Cell Culture starts with methods to prepare of decellularized ECM as a scaffold for 3D cell culture. There are several decellularization methods available – physical or chemical, including detergents and enzymes – and no single protocol is suitable for all tissues. In addition to the need for optimization for each tissue type, the key to choosing the right technique lies in the purpose for which the decellularized ECM will be used. For tissue engineering, it is often desirable to leave the ECM scaffold with preserved architecture and bioreactivity. For bioprinting, preservation of bioreactivity is the priority; destruction of architecture is not an issue because it is custommade by the 3D printing process. As examples of these two different approaches, we present two methods in this book. Chameettachal and Pati provide a protocol for the preparation of decellularized ECM from bovine corneal tissue for the preparation of bioink for bioprinting (Chap. 2). Čimborová and colleagues describe the decellularization of porcine lung tissue to provide a scaffold for lung tissue engineering (Chap. 3).
Following, Chen and Park present a protocol for fabricating a 3D hanging spheroid plate with application in cytotoxicity assays (Chap. 4). The plate allows first spheroid formation, then separation of unbound and dead T cells and tumor cells from the spheroids for easy quantification of the cytotoxic effects. Oliver-Urrutia and colleagues describe the culture of osteoblasts on 3D-printed scaffolds and approaches for analyzing of cellular functions on these scaffolds using several assays (Chap. 5). Their protocols will come handy to researchers in bone tissue engineering field. Biomimetic semisynthetic hydrogels, formed from a combination of star-shaped poly(ethylene glycol) (starPEG) and the glycosaminoglycan, heparin, allow for the 3D culture of various cells and tissues. Holloway and colleagues describe methods for using starPEGheparin hydrogels to study leukemia (Chap. 6).
The second part of the book focuses on protocols for 3D Organoid and Organotypic Cultures. Caruso and colleagues provide a decision tree for the selection of the mammary organoid models (Chap. 7). Sumbal and Sumbalova Koledova describe different 3D co-culture setups of fibroblasts with mammary organoids or spheroids to address specific roles of paracrine signaling and epithelial-stromal contact in mammary epithelial morphogenesis (Chap. 8). Brezak and colleagues present a 3D culture for the differentiation of fibroblasts into adipocytes and a subsequent 3D co-culture method of adipocytes with organoids (Chap. 9). Kuony and colleagues provide a detailed protocol for the isolation of primary embryonic mouse lacrimal gland organoids and mesenchymal cells and their co-culture in 3D Matrigel to create an organotypic culture model of the lacrimal gland (Chap. 10). A 3D organoid culture model of the kidney, established from whole human kidney cells in renal ECM, is described by Chen (Chap. 11). Blazquez and colleagues share their detailed protocol for a macro-metastasis/organ parenchyma interface model, a mechanically supported coculture of brain slice with tumor plague (Chap. 12).
In the third part of the book, entitled Functional Experiments in 3D Cultures, several model systems and approaches are described that allow the study of mechanical properties and/or manipulation of cells and organoids in 3D. Pérez-González and Barbazán describe the culture of cancer-associated fibroblasts and tumor cells on polyacrylamide gels of defined stiffness to measure the mechanical interaction of these cells using laser ablation (Chap. 13). Ginga and Slyman present an organoid perfusion system for modifying luminal contents and apical cell membrane interactions during organoid culture, recapitulating physiological flow within the normally static organoid lumen, and studying the effects of mechanical strain on organoid cell development (Chap. 14). Sgualdino and colleagues describe hiPSC-derived organoids cultured in synthetic PEG hydrogels on a stretching device that allows mechanical actuation of the organoids (Chap. 15).
The fourth part of the book is dedicated to Microfluidic and Bioprinting Approaches for 3D Cell Culture. Here, Yilmaz and Inci describe design and fabrication of a polystyrene microfluidic chip that is further modified in the microchannels with silk fibroin to form a 3D network with MCF-7 cells as a breast cancer cell model (Chap. 16). Aydin and colleagues provide a detailed protocol for fabricating a tumor microenvironment on chip using a microfluidic device with a blood capillary channel, a tumor channel, and a lymphatic channel (Chap. 17). These microfluidic platforms offer attractive alternatives to existing in vitro tumor models for characterizing tumor growth dynamics, evaluating drug treatments, and studying the
underlying mechanisms of drug response in cancer within a physiologically relevant in vitro setting. The method described by Monteiro and colleagues also has applications in in vitro disease modeling and preclinical drug screening, as well as in tissue engineering. It is the method of suspension bioprinting, which executes precise spatial deposition of bioink to create complex tissue and tumor architectures (Chap. 18).
The fifth and final part of the book provides methods for Imaging and Image Analysis of 3D Cell Cultures. Spoerri and colleagues describe time-lapse imaging of 3D embedded spheroids using confocal microscopy and application of mathematical modelling to interpret the experimental data (Chap. 19). Nuernberg and colleagues provide a protocol for whole-mount fluorescence staining and optical clearing of 3D tumor spheroid formation, their 3D in toto confocal microscopy, and subsequent image analysis (Chap. 20).
Thus, with each chapter, the reader will gain a deeper understanding of specific 3D cell culture techniques, including their setup and analysis. It is my hope and belief that this book will inspire further experimentation, from tweaking established protocols to taking bold leaps and pioneering into unexplored territories through interdisciplinary collaborations. Let us drive discovery and innovation in basic and translational research by embracing the complexity of the 3D microenvironment and mimicking the physiological conditions of living tissues.
Acknowledgments
This work was supported by grants from the Grant Agency of Masaryk University (MUNI/G/1775/2020), Ministry of Education, Youth and Sports of the Czech Republic (ERC CZ LL2323 FIBROFORCE), and the Czech Science Foundation (GAČR; GA23-04974S) to Z.S.K.
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Part I
Hydrogels and Scaffolds for 3D Cell Culture
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he only invented this story and told it to the Indescribable in order that, when it came to the point, it might be reasonable (though not necessary) to explain his death as suicide. Then he came, back here and made preparations for his holiday. He was going to take his holiday at Chilthorpe—to be more accurate, he meant to start his holiday at Chilthorpe. He strongly urged Your Lordship to come down and share part of it with him; it was essential to his plan.”
“And that,” suggested the Bishop, “explains his intense eagerness that I should come down?”
“Precisely He made certain, as best he could, that you would arrive here on the morning after him; that you would be told he had gone out to fish the Long Pool, and that you would be asked to follow him. This would ensure that you would be the first witness of his disappearance.”
“His what?”
“His disappearance. He meant to disappear. Not only for the sake of the test, I imagine; he wanted to disappear for the fun of the thing; to see what happened. He wanted to be a celebrity in the newspapers. He wanted to read his own obituaries. That was why he wrote, or rather got Brinkman to write, a letter to the Pullford Examiner, calling him all sorts of names—the letter was signed, of course, with a pseudonym. You found that out, didn’t you, Leyland?”
“Yes, confound it all, I heard only this morning that ‘Brutus’ was really Brinkman. But I never saw the point.”
“Then he sat down and wrote an unfinished letter in answer to these charges. That letter, of course, was to be found after his disappearance, and would be published in thick type by the Pullford Examiner. That would set everybody talking about him, and his obituary notices would be lively reading. He wanted to read them himself. But in order to do that he must disappear.
“The Chilthorpe gorge is a good place to disappear from. Leave your hat on the edge of it, and go and hide somewhere—you will be reported the next morning as a tragic accident. Mottram had made all arrangements for hiding. He was going to spend his holiday incognito somewhere; I think in Ireland, but it may have been on the Continent. He was going to take Brinkman with him. He would disappear, of course, in his car. He had victualled it before he left
Pullford. On his arrival at Chilthorpe his first act was to paint out its number-plate. He hid some notes in the cushions of the car—that, I think, was a mere instinct of secretiveness; there was no need to do so.
“The plan, then, was this. On Tuesday morning, early, Mottram was to set out for the gorge. Almost immediately afterward, Brinkman was to take out the motor, as if to go to Pullford. He was to pick up Mottram, who would hide under the seat or disguise himself or smuggle himself away somehow, and drive like mad for the coast. Later, you, My Lord, would come to the Load of Mischief, and would get the message about going out to join Mottram at the Long Pool. In passing through the gorge, you would (I fancy) have found some traces there—Mottram’s hat, for example, or his fishing-rod; and your first thought would have been that the poor fellow had slipped in. Then, looking round, you would find this letter half-concealed on a high ledge. You would read it, and you would think that Mottram had committed suicide.
“And then—then you would either make the contents of this letter public or you wouldn’t. If Brinkman was right in his estimate, you would keep the letter dark; the death, before long, would be presumed. The Indescribable Company would have been on the point of paying out the half-million when—Mottram would have reappeared, and Your Lordship would have been in a delicate position. If Mottram was right in his view of your character, then you would produce the letter; Mottram’s death would be regarded as suicide, and the Indescribable would refuse all claims. Then Mottram would have reappeared, and would have seen to it that, in one way or another, the Pullford Diocese should be rewarded for the honesty of its Bishop.
“He was not really a very complete conspirator, poor Mottram. He made three bad mistakes, as it proved. Though indeed they would not have mattered, or two of them would not have mattered, if events had proceeded according to plan.
“In the first place, he went and wrote his name in the visitors’ book immediately on arrival. He wanted to leave no doubt that it was Jephthah Mottram in person who arrived at the Load of Mischief on Monday night. He wanted journalists to come down here and look
reverently at the great man’s signature. Of course, in reality, it is a thing nobody ever does on the night of arrival. It has made me suspicious from the very first, as my wife will tell you.
“In the second place, when he took the precaution of drawing up a new will he neglected to sign it overnight. Brinkman, I suppose, pointed out to him that if any fatal accident occurred—say a motor accident—the codicil leaving the half-million to the Bishop would be perfectly valid. To avoid this danger they must have drawn up a new will, and if Mottram had signed this overnight his death would have made it valid. As it was, for some reason—probably because Brinkman himself was drawing it up (I think the writing is Brinkman’s) late on Monday night—the will was never signed and was useless.
“In the third place, he did something overnight which he ought to have left till the next morning. He not only wrote his confidential letter to the Bishop but he went out with Brinkman to the gorge and posted it—put it on the ledge ready for the Bishop to find next morning. He did not mean to go into the gorge at all the next morning. He would start out on the way to it, say, at eight, and at ten minutes past eight Brinkman, driving the car, would pick him up on the road. From the side of the road they could throw over Mottram’s hat, possibly, and they could slide his rod down the rocks, so as to make it appear that he had been there. (Brinkman, in this way, would establish an alibi; he could not be supposed to have murdered Mottram in the gorge.) But it was not safe to let the letter drop in this casual way; therefore the letter must be planted out overnight. There was no great danger of its premature discovery; in any case, Mottram put it rather out of sight on a ledge so high up that only a tall man would see it, and only if he was looking about him carefully.
“That is the complicated part of this business; the rest of it depends on two simple accidents. Mottram went to bed rather early; he was in an excited frame of mind, and determined to steady himself with a sleeping draught. The watch, the studs, were only symptoms of that fussiness we all feel on the eve of a great adventure. I suppose he borrowed a match from Brinkman to light his gas with. But it was a clear night; there was no need of light to go to bed by. But just at the last moment—a fateful moment for himself
—he did light the gas; perhaps he wanted to read a page or two of his novel before turning in.
“The rest of the story could be more easily told upstairs. I wonder if you would mind all coming up into the actual room? It makes it so much easier to construct the scene if you are on the spot.”
The whole party applauded this decision. “This is what is called an object-lesson, in the education of the young,” observed Mr. Pulteney. “The young like it; they are in a position to hack one another’s shins when the teacher’s back is turned.”
When they reached the bedroom, Bredon found himself falling into the attitude of a lecturer. “The guide,” murmured Angela, “taking a party round the ruins of the old dungeon. Scene of the ’orrible crime. Please pay attention, gentlemen!”
“You see how the gas works in here,” began Bredon. “There’s the main tap, we’ll call it A, which controls the whole supply. Tap B is for the bracket; tap C leads through the tube to the standard lamp. It doesn’t matter leaving tap B or tap C on as long as tap A is turned off.
“When Mottram went up to bed, tap B and tap C were both open, but tap A was properly turned off. Mottram took no particular notice of the disposition of taps; he turned on one tap at random, tap A. Then he lit his match, and put it to the bracket, which naturally lit. He then immediately threw the match away. We know that, because we found the match, and it was hardly burned down the stalk at all. Meanwhile, of course, he had also allowed the gas to escape through the tube into the standard lamp; it never occurred to him to light this. The standard was at the other end of the room, close to the open window; the slight escape of gas did not, unfortunately for him, offend his nostrils. Brinkman told me, and it is probably true, that Mottram had not a very keen sense of smell. After a minute or two, feeling ready to go to sleep, he went up to the taps again, and forgot to reverse the process he had gone through before. Instead of turning off the main tap, A, he carelessly turned off tap B. And the light on the bracket obediently went out.
T B
“That is the lesson of a finger-print. Tap A was stiff, and Mottram left a mark when he turned it on; he would have left another if he had turned it off. He did not; he turned off tap B, which works at a mere touch, and of course he left no mark in doing so. There, then, lies Mottram; the sleeping draught has already taken effect; the wind gets up, and blows the window to; tap A is still open, and tap C is still open; and through the burner of the standard lamp the acetylene is pouring into the room.
“Brinkman is not a late sleeper. The Boots, who is the earliest riser in this establishment, tells me that Brinkman was always awake when he went round for the shoes. On Tuesday morning Brinkman must have woken early, to be greeted by a smell of gas. It may have crept in through his window, or even come up through the floor, for the floors here are full of cracks. Once he had satisfied himself that the escape was not in his own room, he must have thought of the room below. When he reached the lower passage, the increasing
smell of gas left him in no doubt. He knocked at Mottram’s door, got no answer, and rushed in, going straight across and opening the window so as to get some air. Then he had time to turn round and see what was on the bed. There was no doubt that he was too late to help.”
“Did he know it was accident?” asked Eames. “Or did he think it was suicide?”
“I think he must have known it was accident. And now, consider his position. Here was Mottram, dead by accident. There up in London was Mottram’s codicil, willing half a million to the Diocese of Pullford. And that codicil had not been meant to become operative. It had been made only for the purposes of the test. And now, through this accident, the codicil, which did not represent Mottram’s real wishes, had suddenly become valid. It would certainly be judged valid, unless—unless the claim were dismissed owing to a verdict of suicide. Brinkman may or may not have been a good man; he was certainly a good secretary. Put yourself in his position, Mr. Eames. He could only give effect to his dead master’s real wishes by pretending that his dead master had committed suicide.
“You remember the remark in ‘The Importance of Being Earnest,’ that to lose one parent may be an accident, to lose both looks like carelessness? So it was with Mottram and the taps. Two taps turned on meant, and would be understood to mean, an accident. But, and this is worth remembering, if all three taps were found on, it would look like suicide. Brinkman acted on the spur of the moment; he was in a hurry, for the atmosphere of the room was still deadly. He wrapped his handkerchief round his fingers, so as to leave no mark, then, in his confusion, he turned the wrong tap! He meant to turn tap B on; instead, he turned tap A off. That sounds impossible, I know. But you will notice that whereas tap A and tap B are turned off when they are at the horizontal, tap C is turned off when it is at the vertical, When Brinkman, then, saw the three taps, B and C were both horizontal, and A was vertical, it was natural, in the flurry of the moment, for him to imagine that if all three taps were in the same position (that is, all horizontal) they would all be turned on. Instinctively, then, he turned tap A from the vertical to the horizontal. And in doing so he left the whole three in the same position in which
they were before Mottram lit his match. No gas was escaping at all. The result of Brinkman’s action was not to corroborate the theory of suicide, but to introduce a quite new theory—that of murder. Halfstifled, he rushed from the room, locked the door on the outside, and took the key away with him up to his room.”
“Steady on,” put in Angela, “why did he lock the door?”
“It may have been only so as to keep the room private till he had thought the thing out, and the Boots may have come round too soon for him. Or, more probably, it was another deliberate effort to encourage the idea of suicide. Anyhow, his actions from that moment onward were perfectly clear-headed. He helped to break down the door, and, while Ferrers was examining the gas, while the Boots was lighting a match, he thrust the key in on the inner side of the door. It was only when he had done this, when he thought that he had made the suicide theory an absolute certainty, that he was suddenly confronted with the horrible mistake he had made in turning the wrong tap. It was a bad moment for him, but fortunately one which excused a certain display of emotion.”
“And he thought he would be run in for the murder?” asked Leyland.
“Not necessarily. But your arrival worried him badly; you got hold of the murder idea from the start.”
“Why didn’t he skip, then? There was the car, all ready provisioned.”
“The trouble is that Brinkman is, according to his lights, an honest man. And he hated the idea of the Euthanasia money going to the Bishop. I was a godsend to him; here was a nice, stupid man, briefed to defend the thesis of suicide. As soon as I came, he tried to take me out for a walk in the gorge.”
“Why in the gorge?” asked the Bishop.
T
“So that I should find the letter. Yesterday he did manage to take me to the gorge, and actually drew my attention to the ledge. I saw a bit of paper there, but it never occurred to me to wonder what it was. Poor Brinkman! He must have thought me an ass!”
“But why didn’t he get the letter himself, and bring it to us? Or leave it lying about?”
“That was the maddening thing, the poor little man just couldn’t reach it. The wind of Monday night had blown it a bit further away, I suspect. Of course, he could have gone out with a step-ladder, or rolled stones up to stand on. But, you see, you were watching him, and I’m pretty sure he knew you were watching him. He thought it best to lead us on, lead me on rather, and make me find out the envelope for myself. When he’d drawn me right across the trail of it, and I’d failed to see it, he was in despair. He decided that he must bolt after all. It was too horrible a position to be here under observation, and fearing arrest at any moment. If he were arrested,
you see, he must either tell a lie, and land himself in suspicion, or tell the truth, and see the Euthanasia money fall into Catholic hands.
“He ordered a car from the garage to meet the train which arrives at Chilthorpe at 8.40. He determined to meet it on the way to the station. I don’t think the thought of the car lying at the garage, with the ‘sangwiches’—I mean the sandwiches—and the whisky on board, occurred to him for a moment. He is an honest man. But on his way to meet the car he would go through the gorge, and make sure that he was followed; he would draw attention to the document, and then disappear from the scene. He had not much luggage; he had only to clear up a few papers, mostly belonging to Mottram. Among these was the unsigned will which had been drawn up, ready for Mottram’s signature, on the Monday night. This he burned; it could be no use to anybody now. He burned it standing at the window, and the last, unburnt piece escaped from his fingers, and fluttered down through a second window into the room below—this room, which had been Mottram’s. That was your find, Leyland. And the odd thing is that it was through this absurd detail that I got onto the track of the whole thing; because one of my patience cards fluttered down through a ground-floor window; and as I was carrying it upstairs I realized that was how the scrap of paper came to be lying about in Mottram’s room. Then I began wondering what the will was, and why Brinkman should have been burning it, and suddenly the whole truth began to sketch itself in my mind, just as I’ve been telling it you.
“Brinkman had bad luck to the last. I dropped that card just after he started out with his despatch-box; he saw that I’d disappeared from the window, and supposed, with delight, that I was following him. With delight, for of course I was the one man who was interested in proving the death to be suicide. He went back to the cache in the gorge, leading me (as he supposed) all the way; then he waited for a flash of lightning, and jumped up so as to draw attention to the envelope. As he came down again he looked round, and, in the last rays of the lightning flash, saw that it was Eames, not I, who was following him. Eames—the one man who would certainly make away with the precious document! But there was no time to be lost; he could hear the taxi already on the hill. He ran round to the
road, leaped on board the taxi, and, in desperation, sent a note to me by the taxi-man telling me to make Eames shew me what he had found. I don’t know where Brinkman is now, but I rather hope he gets clear.”
“Amen to that,” said Leyland; “it would be uncommonly awkward for us if we found him. What on earth could we charge him with? You can’t hang a man for turning the wrong gas-tap by mistake.”
“Poor Mr. Simmonds will be relieved about this,” said Angela.
“By the way,” said the Bishop, “I hear that Mottram did leave some unsettled estate after all, and that, I suppose, will go to Simmonds. Not a great deal, but it’s enough for him to marry on.”
Angela swears that at this point she heard, on the other side of the door, a scuffle and the rustle of departing footsteps. She says you can’t cure maids of their bad habits, really.
“My own difficulty,” said the Bishop, “is about my moral claim to this money. For it was left to me, it seems, by a will which the testator did not mean to take effect.”
“On the other hand, you’ve earned it, My Lord,” suggested Bredon. “After all, poor Mottram was only waiting to find out whether you would prove to be an honest man or not. And I think you’ve come out of the test very well. Besides, you can’t refuse the legacy; it’s in trust for the diocese. I hope Pullford will see a lot of Catholic activity now.”
“The church collections will be beginning to fall off almost at once,” said Eames, with a melancholy face.
“I wish I had scrutinized those motor-cushions more closely,” said Mr. Pulteney. “It seems to me that I get nothing out of all this.”
“Which reminds me,” said Leyland, “I suppose the bet’s off.”
“And Mr. Bredon,” added the Bishop, “will get no thanks from his company. I’m afraid, Mr. Bredon, you will have carried nothing away with you from your visit to these parts.”
“Oh, I don’t know about that,” said Bredon.
Transcriber’s Note
This transcription follows the text of the edition published in 1927 by Jacobsen Publishing Company, Inc. The following changes have been made to correct what are believed to be printer’s errors (based in part on corrections made in later editions).
“injunctions” has been changed to “injections” (Ch. II).
“he ever left” has been changed to “he never left” (Ch. II).
“motion in her voice” has been changed to “emotion in her voice” (Ch. IV).
“swerving to” has been changed to “serving to” (Ch. IV).
“trace it had” has been changed to “trace where it had” (Ch. IV).
“constitutency” has been changed to “constituency” (Ch. VII).
“verybody” has been changed to “everybody” (Ch. XVI).
“put my car in” has been changed to “put my oar in” (Ch. XVIII).
“on the other hand” has been changed to “on the one hand” (Ch. XX, first instance).
“Dr. Ferers” has been changed to “Dr. Ferrers” (Ch. XXIII).
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