Bioengineering & Biomedical Engineering

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skin, intestine and amnion due to lack of structural rigidity. 28 ...... fabrics as artificial skin grafts to experimental burn wounds. J. Biomed. .... Sulkin, M. S. et al.
Bioengineering & Biomedical Engineering.

Bioengineering Final Year Project Report

Exploring the scaffold structure and fluidic properties of organs using the CLARITY and decellularization techniques.

Kokilavani Manokaran 11/05/2016

Supervisor: Dr. Cecile Perrault Dissertation submitted to the University of Sheffield in partial fulfilment of the requirements for the degree of Bachelor of Engineering.

Acknowledgement I have enjoyed my time during this project a lot, and I’d like to thank each and every individual who made this experience as great as possible for me. I’d like to sincerely thank my supervisor Dr. Cecile Perrault for her extraordinary support throughout the time of this project and for being a wonderful facilitator, helping me with the project and making me feel appreciated at every single point. I’d cherish this amazing experience for the rest of my life and thanks to all the little talks we’ve had on academic and non-academic topics. I’d also like to thank our lab manager Julie Marshall for helping everyone in the lab and managing the lab. And sincere thanks to all the wonderful Ph.D. students, Claudia Wittowskie, Robert Owen, Dirar, Aban, and everyone else who were always there to help undergraduates whenever they needed any help. Thanks to Raj Kampa for his help and support throughout this project. I’d especially like to thank my Ph.D. support Luke Boldock for letting me use all his lab equipment and guiding me with the lab etiquettes and also getting me organ samples for my experiment and thanks to Marwa Mahmoud for providing me with the samples. I cannot express my gratitude in words, and I’d like to thank all of you once again for the wonderful time I had working with every single one of you.

I would finally like to thank and dedicate all my work to my mother and my aunt who have supported and raised me throughout tough times, and I wish to be a good friend to someone like they have been to each other. I cannot thank them enough for being there always as my family and my friends. And thanks to all my friends for being the best that they can be and supporting me and letting me be who I am and appreciating me that way. I once again would like to thank everyone and I wish all a happy and healthy life.

Table of Contents Acknowledgement ...................................................................................................................... i Abstract ...................................................................................................................................... 3 1.

2.

Introduction ........................................................................................................................ 5 1.1.

About ........................................................................................................................... 6

1.2.

Aims and objectives .................................................................................................... 6

1.3.

Outline of the project................................................................................................... 7

1.4.

Decellularization ......................................................................................................... 7

1.5.

CLARITY.................................................................................................................... 7

1.6.

Project timeline ........................................................................................................... 8

Literature Review ............................................................................................................... 9 2.1.

Brief........................................................................................................................... 10

2.2.

Decellularization ....................................................................................................... 10

2.2.1.

Introduction ........................................................................................................... 10

2.2.2.

Decellularization and clinical challenges .............................................................. 11

2.2.3.

Decellularization for the generation of ECM scaffolds ......................................... 12

2.2.4.

Chemical Compounds............................................................................................ 13

2.2.5.

Biological Solutions .............................................................................................. 14

2.2.6.

Other Agents .......................................................................................................... 14

2.2.7.

Decellularized organ matrices: what’s left behind? .............................................. 15

2.2.7.1.

Defining decellularization .................................................................................. 15

2.2.7.2.

The effect of decellularization on ECM composition ........................................ 15

2.2.7.3.

The effect of decellularization on ECM structure.............................................. 16

2.3.

CLARITY.................................................................................................................. 17

2.3.1.

Introduction ........................................................................................................... 17

2.3.2.

Purpose of CLARITY ............................................................................................ 17

2.3.3.

Obtaining clarified tissues ..................................................................................... 19

2.4.

Imaging techniques ................................................................................................... 21

3.

Materials and methods ...................................................................................................... 25

4.

Results .............................................................................................................................. 29

5.

Discussions/Conclusion .................................................................................................... 41 5.1.

Decellularization ....................................................................................................... 42

5.2.

CLARITY.................................................................................................................. 43

5.3.

Decellularization and CLARITY .............................................................................. 44

6.

Abbreviations.................................................................................................................... 47

7.

References ........................................................................................................................ 49 2

Abstract The biomechanics and tissue structures of whole organs have remained superficial for decades. Cell staining has been used to obtain cell orientation images for the past few decades and recent developments in the past couple of decades include highly technological microscopes like scanning electron microscopes, confocal and fluorescent microscopes and second harmonic generation microscopes give better details of the cell matrix. Decellularization of an organ helps one to understand the ECM in-depth, and one can also understand the vasculature of an organ after decellularization has been performed. But to be able to map the alignment of cells in organ within the matrix, one has to remove all the lipid bilayer membranes present in an organ as it gives the opacity to the tissue. Lipid bilayers are removed by a procedure called CLARITY and using this procedure hydrogel crosslinked structure can be obtained from a whole organ sample without distorting the structure and mechanical properties as cells are embedded in a hydrogel allowing to preserve all the fine structures within the tissue in cellular level. In this report, mice organs have been analysed deeply to get an overview on the effect of decellularization and CLARITY individually and have also pioneered performing both the procedures on the same organ to see the effects on the resolution images and the quality of ECM. Results show distinctive properties upon the performance of each procedure and against expected believes, performing both procedures together gives an ECM embedded with hydrogel rather than a structure-less matrix.

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List of figures: Figure 1: Decellularization and recellularization. .................................................................... 11 Figure 2: Decellularized organ scaffolds. ................................................................................ 13 Figure 3: CLARITY procedure. ............................................................................................... 18 Figure 4: Lipid bilayer structure. ............................................................................................. 20 Figure 5: Fluorescence microscopy. ........................................................................................ 22 Figure 6: Confocal microscopy. ............................................................................................... 23 Figure 7: Light sheet microscopy. ........................................................................................... 24 Figure 8: Decellularization - Kidney 1 ....................................................................................30 Figure 8: Decellularization - Kidney 2 ....................................................................................31 Figure 8: Decellularization - Liver 1 .......................................................................................32 Figure 8: CLARITY - Kidney 1 ..............................................................................................33 Figure 8: CLARITY - Kidney 2 ..............................................................................................34 Figure 8: CLARITY - Liver 1 .................................................................................................35 Figure 8: CLARITY - Liver 2 .................................................................................................36 Figure 8: Decellularization and CLARITY - Kidney 1 ..........................................................37 Figure 8: Decellularization and CLARITY- Kidney 2 ...........................................................38 Figure 8: Decellularization and CLARITY - Liver 1 .............................................................39 Figure 8: Comparison table.....................................................................................................40

List of Tables: Table 1: Required materials for decellularization .................................................................... 26 Table 2: Hydrogel solution – materials required ..................................................................... 28 Table 3: Clearing solution – materials required ....................................................................... 28

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1. Introduction

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1.1.

About

Understanding of tissue ultrastructure and scaffold properties is crucial to understand the complexity or human system at a cellular and molecular level. Current major researches in the field of biomedical research include 3D cell culture, organ on a chip, nerve regeneration and much more and there is a lack of details of cells and scaffolds at their original state. Throughout times tissue structures have been understood at a planar microscopic level, which fails to provide 3-dimensional details of cell orientation and cell alignment in the extracellular matrix. Addressing this concern, this project involves two distinct techniques that will help one to deeply understand the three-dimensional alignment of cells in mouse liver and kidney. Using decellularization1, an ECM scaffold is obtained which serves as a control for scaffold investigation. Using CLARITY2, a transparent organ is obtained which serves as a control for 3D dimensional analysis for cell structure and embedding. Further exploration of these two techniques together in one organ provides a platform to understand the properties of an organ from different aspects of its structure including the role of the lipid bilayer in tissue integrity, vasculature and cell embedment in an ECM scaffold.

1.2.

Aims and objectives

The key aim of this project is to perform 3D imaging on a decellularized scaffold and clarified sample obtained using CLARITY procedure and to explore the basic scaffold structure on mouse liver and kidney samples, without disrupting the mechanical properties of the tissues and also making sure the cellular structures of the organs remain intact at the same time. The key objectives are: 

To decellularize whole liver and kidney samples to obtain an extra-cellular matrix (ECM) scaffold



To perform CLARITY on whole liver and kidney samples to make them transparent



To obtain high-resolution images of both decellularized and clarified organ samples using fluorescence or confocal microscope to analyse the ultra-structures of the samples



To perform decellularization and CLARITY on the same organ



To characterize the scaffold and obtain high-resolution images of the same scaffold



To demonstrate the biomechanics of all three scaffolds by analysing fluidic properties

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1.3.

Outline of the project

The purpose of the project is to obtain three scaffolds with distinct characteristics to be able to provide details on the cells and scaffold structure of the organs. This aim is to be achieved by decellularizing an organ and performing CLARITY on an organ followed by decellularization and CLARITY performed on the same organ. The results of these three procedures would be distinct from each other. The scaffolds will then be used to analyse cell orientation and embedding, ECM structure and clarified ECM structure of the organs in detail using cell staining and fluorescence or confocal microscopy.

1.4.

Decellularization

In the year 20061, Stephen F. Badylak pioneered the procedure of decellularization at the McGowan Institute for Regenerative Medicine at the University of Pittsburgh1. Decellularization of an organ involves using physical, chemical and mechanical stimuli to remove cells from the tissue. By applying these stimuli to a fresh tissue, one can obtain an ECM scaffold. ECM scaffolds can be later used to replace diseased or damaged organ of a patient by recellularizing the ECM scaffold with function-specific cells obtained from the patient.

1.5.

CLARITY

CLARITY is a technique developed in the Deisseroth lab at Stanford University in 2010 by Dr. Karl Deisseroth and his team. CLARITY stands for Clear Lipid-exchanged Anatomically Rigid Imaging/Immunostaining-compatible Tissue hYdrogel3. CLARITY can be used to clear lipid bilayer membranes from tissues and by clearing the tissue while preserving fine structural details, CLARITY provides a technique for obtaining high-resolution information from complex systems while maintaining the global perspective necessary to understand system function.

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1.6.

Project timeline

Decellularization PBS wash

0.01% SDS

0.1% SDS

1% SDS

dH2O + Triton X100

PBS

Hydrogel perfusion

Polymerization

Clearing

Immunostaining

Imaging

CLARITY Monomer solution

Decellularization and CLARITY Perform Step 1-6

Decellularization

Obtain ECM

Perform Step 1-6 CLARITY

Obtain decellularized and clarified organ

Imaging

The overall time it takes for decellularization is about a week and the time it takes for CLARITY depends on the size and type of the tissue, and it varies from two weeks to few months.

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2. Literature Review

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2.1.

Brief

Both decellularization and CLARITY are quite recent establishments in the field of biomedical engineering, and they both have claimed a vast amount of interest around the world for their uniqueness and versatility. Decellularization has been in research for more than a decade and has been performed on different organs whereas CLARITY has not been performed on any other organs than mouse brain which leaves a vast opportunity to explore other organ structures. In this project, mice livers and kidneys have been used, and their structural and biomechanical properties have been studied in depth.

2.2.

Decellularization

2.2.1. Introduction Traditional organ transplantation needs an effective alternative to increasing the number of organ transplantations and the success rate of the surgeries. An alternative robust enough to produce long-term outcomes and considerably decrease the number of patients on the waiting lists every year. For the past few years, studies have been conducted on whole-organ tissue engineering extensively to address the needs for a better option to traditional transplantation methods. It has been 30 years since the very first investigation of skin tissue engineering and the progress made afterwards in engineering various tissues including cartilage, bladder, skin and cornea has been remarkable4,5,6. However, development of organs has stayed at a tissue level in the past due to lack of scaffolds with 3D complexity equivalent to that of host tissue and organ inducing a course of research in the field of whole organ engineering, and this has been achieved so far using the decellularization technique.

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Figure 1: Decellularization and recellularization. A simple schematic to show the underlying concept of decellularization and recellularization that are bein vastly used in the field of biomaterials and tissue engineering7.

Decellularization can be defined as a procedure used to remove cells from the extracellular matrix (ECM) of an organ to construct a three-dimensional organ scaffold with the same structural integrity as the host tissue8,9,1. First in the late 80s, several instances were reported on decellularization of simple tissues and small sections of organs. However, in the early 2000s, the decellularization procedure was used to generate a whole heart scaffold successfully. Shortly after that whole organ decellularization was being performed using similar approaches on various organs like pancreas, liver, lungs, kidneys and intestines and species including primates, rodents, porcine and human10,11. 2.2.2. Decellularization and clinical challenges

Before this development, the task of creating a whole organ scaffold with native composition and intricate architecture required to engineer organs that are functional was very challenging12. Further research was done on analyzing the capabilities of the scaffold and to establish the potential of these scaffolds, cells of specific functionality related to the decellularized tissue were seeded onto the scaffold and various studies have thus far reported clinical functionality of the recellularized tissue in small animal models 13. Despite notable progress, challenges with significant concerns still exist as there haven’t been notable records of successful experiments on human level organs. Finding the right cell type for specific 11

organs for recellularization is clinically challenging and ultimately restoring vasculature and scaffold structure after the procedures are performed for long-term function after transplantation has been an important issue as well14,15,16.

2.2.3. Decellularization for the generation of ECM scaffolds Decellularization aims at providing a biological scaffold for transplants without any adverse effects on the biomolecular composition, structural integrity and biomechanical properties of the ECM after lyzing all the molecular and nuclear materials efficiently. Decellularization processing step intended to eliminate cell from the tissue will affect the three-dimensional architecture of the ECM scaffold. Therefore, requiring a need to optimize decellularization protocols for different tissues to avoid damage to native ECM structure. Various protocols are used for decellularization of tissues, and they mostly comprise a combination of physical, chemical and biological (enzymatic) approaches 17. Over the time, different protocols have been developed with employ combinations of these factors on native as well as engineered tissues to successfully remove cellular materials without disrupting the ECM structure. The initial process of decellularization involves utilization of physical treatment or chemical solutions to lyze the cell membrane and then separating cellular materials from the ECM using enzymes and reagents18. The agents cause solubilisation of cytoplasmic elements and cellular materials, and the final step involves removal of cell remains. Mechanical agitation is known to enhance the process of decellularization. It is required to remove all the residual detergents and reagents after the procedure due the risk of an adverse host response to the chemicals used. Several of these techniques are combined to improve the feasibility of the procedure by reducing possible unfavourable conditions 19,20.

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Figure 2: Decellularized organ scaffolds. Various mouse organ scaffolds shown in a comparative manner to the host tissue and ECM scaffold after decellularization. The absence of any colour represents the lack of blood and host cells in the tissue 21.

2.2.4. Chemical Compounds

Most prevalent methods of decellularization use chemical compounds to remove cells from the tissue. Commonly used chemical reagents are acids, bases, alcohol, acetones, glycerol, hypertonic solutions and hypotonic solutions, ionic and non-ionic detergents. Each of these compounds acts differently on native tissue and decellularize the tissue. Acids and bases 22,23 mostly work on proteins and are responsible for denaturing proteins and also responsible for modifying nucleic acid and solubilising cell components leading to cell rupture. However, the nature of the reagents are non-selective and may destruct crucial elements of ECM such as collagen, glycosaminoglycans, growth factors and other elements. Hypertonic and Hypotonic12 solutions use the principle of an osmotic shock to disrupt the cells and also cause interference between DNA and protein interactions. These solutions are very effective in disrupting cells but are not effective in removing cellular remains. A combination of ionic and non-ionic detergents13 or each of them individually can disrupt interactions between DNA and protein, and may destroy lipids, lipoproteins and protein molecules, thus leading to the destruction of ECM structure and deterioration of scaffold materials.

Dehydration and cell lysis are caused by alcohol and glycerol. On the other hand, acetone can be employed in lipid removal in decellularization16. But, both acetone and alcohol tend to fixate tissues causing damages to the ECM making the scaffold less viable for clinical 13

use24,14. Organic solvent like tributyl phosphate (TBP) seems more efficient in certain tissues such as tendons, but less efficient compared to Triton X-100 and sodium dodecyl sulphate (SDS) in soft tissue decellularization25.

2.2.5. Biological Solutions

Enzymatic agents can be used to specifically target nucleic acids and peptide bonds during decellularization in an attempt to extract cells from the ECM. But enzymes have a capability of remaining attached to ECM components which might cause adverse reactions at a higher level26. Long-term exposure may also lead to the destruction of whole ECM components at a considerable level. Nonenzymatic agents such as ethylene glycol tetraacetic acid and ethylenediaminetetraacetic acid (EDTA) accumulate with ions and metal ions that promote cellular materials to detach from ECM proteins15.

2.2.6. Other Agents

Physical factors like pressure, force, temperature and nonthermal permanent electroporation (NTIRE) are used in decellularization procedures 27. Tissue samples can be exposed to extreme temperature cycle can be used to carefully remove cells without loss of ECM proteins. Surface tissue decellularization can be easily performed using a combination of mechanical, chemical, enzymatic, non-ionic and hypertonic agents in organs like bladder, skin, intestine and amnion due to lack of structural rigidity28. Electroporation methods have been scrutinized as an approach to decellularization of tissues. The optimal environment for decellularization agents is dependent on characteristics of the tissue such as thickness, the reagents being used, density and the clinical function of the decellularized tissue. Before applying the agents, any unwanted tissue can be reduced to simplify the cell removal process29. Currently, the most common issues being dealt with regarding decellularization are a dependency on cell disruption, the excess cell remains causing immune reactions when absorbed into ECM and maintenance of tissue structure and other components after the procedure. And all these issues remain unresolved despite the choice of different agents25.

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2.2.7. Decellularized organ matrices: what’s left behind? 2.2.7.1.

Defining decellularization

A lot of decellularization protocols exist for different purposes and due to the variation in decellularization methods choosing the most favourable decellularization method is somewhat inscrutable. However, with further research, the feasibility of whole organ decellularization is indisputable. The key areas for comparison of decellularization procedures are the efficiency of cell removal and the capability of ECM preservation. It is suggested that cell removals be quantified visually using hematoxylin and eosin (H&E) or DAPI staining. “The aim is to have around 50 ng dsDNA/mg* tissue in dry weight remaining after decellularization; also, the fragment length of the DNA should be around 200bp*.”30 Following this guidance, the optimal protocol for particular tissue can be assessed and used for clinically relevant performance31.

2.2.7.2.

The effect of decellularization on ECM composition

It is very crucial to analyse the quality of the ECM obtained after decellularization as it has an impact on the clinical functionality of the organ after recellularization. Partial decellularization or over usage of reagents may both lead to incapability of the scaffold to perform in a clinical environment. To ensure the viability of the scaffold after decellularization, it must be investigated for structural integrity, ECM architecture and microvasculature22,32,33. Although decellularization procedure maintains the integrity of scaffold it also it one of the biggest concerns regarding this procedure as the structural integrity is not quantifiable. Retention of scaffold materials like collagen, laminin, fibronectin and elastin have been reported by many and depletion of ECM proteins has also been reported. One group has reported details on the influence of various reagents on lung decellularization. SDS used to decellularize lung affected elastin and collagen level to a greater degree than a zwitterionic detergent, but both agents reduced GAG content considerably34,30. Another study compared multiple protocols and reagents on the effectiveness of decellularizing rat hearts and reported that none of the procedures and reagents provided a favourable and viable result. And the same study has reported instances of the cell remains not being cleared by detergents. Contrarily when cell remains are removed, ECM structures

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are deteriorated causing a bigger concern35,36. Triton X-100 is very effective in retaining scaffold properties but does not remove cells effectively in kidney decellularization procedures. All these studies prove that there is no ideal protocol available and figuring out the needs and reagents suitable is crucial in preserving the ECM components 37.

2.2.7.3.

The effect of decellularization on ECM structure

The preservation of ECM components leads to the preservation of mechanical properties and ultrastructure of the scaffold, which may aid in recellularization. Various reports have demonstrated the structural integrity of parenchymal structures such as bile duct, tricuspid valve, glomeruli, alveoli, myocardial and epicardial fibres 30. The maintenance of the complete vasculature of ECM is important alongside the structural integrity of ultrastructures for successive recellularization of organ scaffolds afterwards 6. Studies have reported preservation of complete vasculature when liver was decellularized with 1% Triton X-100, whereas 1% SDS decellularization destroyed vasculature in the organ. But, the same concentration of reagents showed the opposite reaction on a lung decellularization procedure38. Again denoting that the feasibility of protocol must be determined previously based on the tissue type to be able to preserve ECM structure. One more factor that counts to optimal decellularization protocol is the method of delivering reagents to the tissue39. Passive delivery of reagents through native vasculature may be good but not in all case. Perfusion decellularization is preferred but with extensive care towards flow rate and complications that arise with it.

2.2.7.4.

Effects of decellularization on imaging and need for CLARITY

Decellularization allows characterization through various imaging techniques. Recellularized tissue scaffolds almost have the same properties as the host tissue, and high-resolution imaging of whole organs is not very feasible. Various microscopic imaging analyses performed on the host as well as decellularized and recellularized tissues lack to provide ultrastructural details of cell structures and vasculature. In this regard, a better technique is required to map the arrangement of cells in their native state in high resolution and obtain three-dimensional images40.

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2.3.

CLARITY

2.3.1. Introduction

CLARITY is a technique recently developed for the purpose of mapping nervous system. The method is used to transform an intact tissue such as whole organs into an optically clear and permeable hydrogel-embedded form that can undergo immunostaining and high-resolution three-dimensional imaging without damage to the sample3. By clearing the tissue while preserving fine structural details, CLARITY offers a technique for obtaining high-resolution information from complex systems while preserving the global aspect necessary to understand system function41.

2.3.2. Purpose of CLARITY

The complexity of obtaining comprehensive structural and molecular information from intact tissues has been an obstacle in studying complete biological systems. Extensive structural analyses of whole organs, not reconstructed tissue sections and molecular phenotyping are considered necessary to gain a full understanding of the relationships and functional mechanisms of biological systems42. However, achieving both types of analysis in intact tissues has been proven to be difficult with traditional approaches. Techniques that support intact tissue imaging and structural analysis are unsuitable for molecular phenotyping while techniques that support molecular labelling require thin tissue sectioning which restricts structural reconstruction43. CLARITY addresses the need for studying complicated systems intact with molecular-level resolution and also supports molecular phenotyping and tissue imaging of intact tissues44.

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Figure 3: CLARITY procedure. A pictorial representation of the whole CLARITY procedure to provide an idea of basic concepts and presumptions behind the working of the technique45.

CLARITY is intended to present three-dimensional images of long-range intricate cellular projections for mapping of tissue structure and provides visualization of a variety of tissue types. Up till now, CLARITY has shown to be a feasible procedure for imaging of whole organs without the need for sectioning and facilitates imaging of long-range projections, local inter-linking wiring, intercellular and intracellular connections, sub-cellular structures, nucleic acids, protein complexes and neurotransmitters. Whole organ tissues processed with CLARITY have also shown feasibility with in situ hybridization46. CLARITY also allows antibody labelling techniques and supports multiple rounds of staining and de-staining47.

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2.3.3. Obtaining clarified tissues

The major obstacle to observing intact tissues and organs is the presence of protective lipid bilayers within the tissue. The lipid membranes excessively limit the accessibility of molecular probes into the tissue by creating a diffusion barrier. And also, the scattering of light at the lipid-aqueous interface causes the tissues to be opaque and restricts the imaging depth which makes it more suitable for light microscopy2. However, the lipid bilayer provides the structural integrity, and biomolecular retention in the tissue and removal of it would result in causing damage to tissue and extensive loss of cellular materials. That is where CLARITY offers a way to securely retain the biomolecules even after removing lipid bilayers by attaching the biomolecules to a hydrogel matrix first to preserve structural integrity and then washing the unattached lipids afterwards. As a result, a clarified tissuehydrogel hybrid is obtained with biomolecular details intact which is optically transparent and permeable for macromolecules. With the lipids being removed from the tissue, penetration of light and macromolecules becomes easier making it ideal for molecular phenotyping and three-dimensional imaging without the need for tissue sectioning48–51.

To create a hydrogel matrix within the whole organ, hydrogel monomers including acrylamide and formaldehyde is infused into the tissue at low temperature. Biomolecules such as nucleic acid, proteins, and small molecules within the tissue, are linked covalently to the acrylamide monomers in reaction with formaldehyde52. After hydrogel monomer infusion using a thermal initiator, the acrylamide polymerization can be triggered during hightemperature incubation to form a hydrogel network crosslinked and held intact by the attached biomolecules and a small chemical crosslinker. As a result, a hydrogel-embedded tissue is left which contains all the macromolecules crosslinked to the hydrogel and lipid bilayers are left behind as they lack the functional group required for chemical reaction with the hydrogel monomers53.

After obtaining hydrogel embedded tissue network, the unattached lipid bilayer membranes can be washed away without disrupting the tissue structure or causing any damage. An ionic detergent preferably sodium dodecyl sulphate (SDS) can be used to remove lipid membranes from the mesh as SDS forms micelles in aqueous solutions. Thus, leaving the biomolecules crossed linked to the hydrogel matrix54.

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2.3.4. Effects of lipid bilayer on high-resolution imaging High-resolution images are generally obtained using laser aided microscopes like confocal microscopes, or fluorescent microscopes can also be used for that purpose. Lasers lack the ability to trace tissues past the boundaries of interlaying lipid bilayer membranes.

Figure 4: Lipid bilayer structure. Structural representation of liposome, micelle and bilayer sheet. The structure of micelle and bilayer comparatively explains the hypothesis of CLARITY55.

2.3.5. Immunostaining

Immunostaining is required to image clarified tissues to stain the cellular materials and other scaffold substances to visualize the tissue under microscopes. Primary and secondary antibodies like Parvalbumin, Tyrosine Hydroxylase, DAPI, Propidium Iodide and Neurofilament NF-H can be used and fluorescent dyes like fluorescein can also be used for this purpose56.

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2.4.

Imaging techniques

2.4.1. Confocal microscopy to obtain 3D images of clarified tissues Clarified tissues should be imaged using a microscopy technique that focuses on a single plane at a time within the sample. Standard light microscopy will not be appropriate for the optically transparent and fluorescent stained whole tissue samples as light will pass through the entire sample and fluorescence will be emitted from everywhere. As a result, the signal from the focused plane will be diluted and lost by the signal directed from other regions in the sample, causing the sample to resemble a globule of autofluorescence under the light microscope. To obtain high-resolution image, it’s preferable to use confocal microscope, but fluorescence microscope and light sheet microscopes are very useful as well 57,58.

Image analysis

Two-dimensional analysis of clarified tissues can be performed using open source Java platforms such as ImageJ and Fiji. Three-dimensional image analysis of clarified tissue samples can be performed using any open source (free) resources (Vaa 3D and Micro Manager) or commercially available imaging software (Metamorph, Imaris, and Amira). Most software are compatible with files from the majority of confocal microscopes. They mostly vary in their user interface and ease of use alongside the price.

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2.4.2. Fluorescence microscopy Fluorescence microscopy can be used to obtain images. Samples must be dyed with fluorescent dyes and are widely available. Obtaining complex 3D rendered images may not be possible with all fluorescence microscopes, but it can still be used for CLARITY based tissue imaging50.

Figure 5: Fluorescence microscope. A sectioned image of a 3D modelled fluorescence microscope that shows the interior alignments of various systems and parts involved 59.

Advantages 

Commonly used in laboratories



Most cost efficient out of all the other options



Easy to use and fast processing time

Disadvantages 

Three-dimensional imaging is hard

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2.4.3. Confocal microscopy Clarified tissue samples can be imaged using standard confocal microscopy. Objectives with long working distances and a high number of aperture will be desired for maximum imaging depth and high resolution50.

Figure 6: Confocal microscope60. A graphical representation of a confocal microscope’s working model which shows the placement of lasers, filters, and detectors on the microscope.

Advantages 

Most commonly used microscopy technique



Supports image tiling to cover up large areas of tissue



No specific modifications needed for CLARITY purposes, just optimal objectives

Disadvantages 

Slow processing time, especially for high resolution and large tissue sections



Illuminates entire sample during imaging - leads to photobleaching after a couple of days

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2.4.4. Light sheet microscopy (SPIM) Light-sheet microscopy is

another three-dimensional imaging technique

although

complicated can be used for imaging clarified samples. However, the currently available light sheet systems are designed for very small tissue samples and may not be very efficient in imaging bigger clarified tissues.

Figure 7: Light sheet microscope61. Working schematic of a selective plane illumination microscopy (SPIM) showing the vertically positioned detectors and cameras.

Advantages 

Fast processing times



No sample photobleaching



Resolution better than confocal microscopy

Disadvantages 

Not widely used and very expensive



Thickness of light sheets is very thin making it harder to image bigger samples

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3. Materials and methods

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Decellularization protocol62

3.1.

Tissue Mice tissue obtained from the medical school. Breed/Stain: C57BL/6 – Black inbred female mice Procedure17,62

1. Wash the liver with Phosphate Buffered Saline solution overnight through perfusion via portal vein at 6 ml/min. 2. Perfuse the liver at 6 ml/min with Sodium Dodecyl Sulphate (SDS) in distilled H2O for 24 hours with 0.01% (wt/v) SDS 3. Perfuse the liver with SDS in distilled H2O for 24 hours with 0.1% (wt/v) SDS 4. Perfuse the liver with SDS in distilled H2O for 24 hours with 1% (wt/v) SDS 5. Afterwards, wash the liver with distilled H2O for 15 mins and with 1% (wt/v) Triton X – 100 for 30 mins to remove residual SDS. 6. Wash the liver with PBS for 1 hour. Decellularization - Required materials and suppliers63 Ingredient

Required

PBS

100ml

10X

SDS

10g

0.01%

Supplier (CAT.No)

Cost

BP3991-1L

£54.70

- L3771-500G

£226.50

1% Triton X-100

5ml

T8787 – 100ml

1%

£27.20

Table 1: Required materials for decellularization 3.2.

CLARITY protocol2

Solution Preparation 1. Make 1 L 10x 0.1 M phosphate buffer saline (PBS, pH 7.4): Weigh 26.2 g sodium phosphate (monobasic), 115 g sodium phosphate (dibasic), and 87.66 g NaCl. Dissolve in about 800 ml distilled H2O, adjust pH, and bring the volume up to 1 L with distilled H2O.

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2.

Make 1 L 0.1 M phosphate buffer saline (pH 7.4): Take 100 ml 10x PBS, and add 900 ml distilled H2O.

3. Make 1 L 1x 0.1 M phosphate buffer saline (pH 7.4) with 0.1% Triton X-100: Take 100 ml 10x PBS, add 1 ml Triton X-100, and bring the volume up to 1 L with distilled H2O. 4. Make 1 L 10x 0.1M phosphate buffer (PB, pH 7.4): Weigh 26.2 g sodium phosphate (monobasic) and 115 g sodium phosphate (dibasic). Dissolve in about 800 ml distilled H2O, adjust pH, and bring the volume up to 1 L with distilled H2O. 5. Make 1 L 0.1 M phosphate buffer (pH 7.4): Take 100 ml 10x PB, and add 900 ml distilled H2O. 6. Make 1 L 0.1 M phosphate buffer (pH 7.4) with 0.1% Triton X-100: Take 100 ml 10x PB, add 1 ml Triton X-100, and bring the volume up to 1 L with distilled H2O. 7. Make 400 ml hydrogel monomer solution (4% acrylamide, 0.25% bis-acrylamide, 4% paraformaldehyde, 0.0075% ammonium persulfate, 0.0005% saponin in 0.1 M PBS): Weigh 0.3 g ammonium persulfate and 0.2 g saponin. Add 210 ml distilled H2O, 40 ml 40% acrylamide, 10 ml 2% bis-acrylamide, 40 ml 10x PBS (pH 7.4), 100 ml 16% paraformaldehyde, and mix well. 8. Make 4 L clearing solution (4% SDS in 0.2 M boric acid): Weigh 49.464 g boric acid and 160 g SDS. Dissolve in about 3.5 L distilled H2O, adjust pH to 8.5 with NaOH, and bring the volume up to 4 L. 9. Make 1 L 80% glycerol solution: Measure 800 ml glycerol, add 200 ml distilled H2O and mix well. Tissue Preparation2,3,64

1. Prepare hydrogel monomer solution and place it the centrifuge tube. 2. Take the whole tissue and perfuse the hydrogel for two days at 4 °C. Make sure that the hydrogel solution is at least 10x the volume of the tissue (i.e.,20mlx10=200ml) 3. Clear the perfusion tubing with distilled water to avoid polymerization resulting in blockage. 4. Bring the hydrogel fixed tissue to a fume hood. 5. Polymerize the hydrogel fixed tissue by adding 2ml TEMED, mix well, and let sit in the fume hood at room temperature for 2-3 hr.

27

6. Remove the excess gel thoroughly. Note: Extra gel will hinder the clearing process. 7. Incubate the fixed tissue in 100 ml clearing solution in an incubator shaker set at 70 rpm and 55 °C for several days until the tissue becomes transparent. 8. Change clearing solution at least once per day and remove the excess gel from the tissue when changing the clearing solution. (Could try perfusing the clearing solution but the incubation temperature must be kept in mind.) 9.

Rinse the tissue in 100 ml 0.1 M PB with 0.1% Triton X-100 at 70 rpm and 37 °C overnight. Repeat this step 3 times and remove the excess gel from the tissue when changing the buffer solution.

10. Incubate the clarified tissue in 80% glycerol at 70 rpm and 37 °C overnight.

3.3.

CLARITY materials and suppliers

Hydrogel Solution (400mL) Ingredient

Required

Acrylamide

40 ml

Supplier (CAT.No) 4%

40%

(40%) Bis (2%)

Cost

Acrylamide £77.00

Solution #161-0140 10 ml

0.05%

2%

Bis

Solution £70.00

#161-0142 VA-044

1g

0.25%

Initiator

Wako Chemicals 2,2 £41.00 -AZOBISY2-(2IMIDAZOLIN-2

Saponin

2g

0.3%

47036-50G-F

£34.70

Table 2: Hydrogel solution – materials required Clearing Solution (4L) Ingredient

Required

Supplier (CAT.No)

Boric Acid

123.66g

200mM

B7901-1KG

£89.30

SDS

400g

4%

L3771-500G

£226.50

NaOH

To pH 8.5

-

72068 – 100ml

£50.90

Table 3: Clearing solution – materials required

28

Cost

4. Results

29

Decellularization Following are the results obtained using fluorescence microscopy on decellularized scaffolds. These images clearly show the structure of ECM scaffold after decellularization. Sample 1 – Kidney Figure 8a shows the ECM structure, and it’s evident from the picture that there are no cells present in the decellularized scaffold. Figure 8b shows a closer look at the ECM matrix and slightly disrupted vasculature can be seen.

Figure 8: Left to right; a) 10X magnification image of a decellularized kidney ECM scaffold; b) 40X magnification image of the same ECM scaffold stained with DAPI and imaged under fluorescence microscope shows the absence of cells in the decellularized matrix.

30

Sample 2 – Kidney Figure 9a shows the ECM structure and evidently there are no cells present in the decellularized scaffold. Figure 9b shows a closer look at the ECM matrix and slightly disrupted vasculature can be seen.

Figure 9: Left to right; a) 10X magnification image of a decellularized kidney ECM scaffold; b) 20X magnification image of the same ECM scaffold stained with DAPI and imaged under fluorescence microscope shows the absence of cells in the decellularized matrix.

31

Sample 3 – Liver Figure10a shows intact microvasculature of a decellularized liver scaffold and figure 10b is a closer look at the intact ECM again proving the absence of any cells in the scaffold viewed at 40X magnification.

Figure 10: Left to right; a) A 4X magnification image of a decellularized liver scaffold stained with fluorescein and imaged under fluorescence microscope clearly showing the vasculature of the ECM scaffold; b) A 20X magnification image of the same sample shows the scaffold with voids clearly indicating missing hepatocytes.

32

CLARITY These fluorescence microscopic images are of clarified livers and tissues showing the cell structures embedded in the hydrogel. Sample 1 – Kidney Figure 11a reveals a hydrogel embedded kidney tissue where one can clearly see the circular shaped cells embedded in the tissue and figure 11b shows a closer look at the embedded tissue.

Figure 11: Left to right; a) A 20X magnification image of a clarified kidney stained with DAPI and imaged under fluorescence microscope clearly showing the cells embedded along the curvature or gluomeruli structures ; b) A 40X magnification image of the same sample gives a closer look at the alignment of cells within the organ.

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Sample 2 – Kidney Figure 12a reveals a hydrogel embedded kidney tissue where one can clearly see the circular shaped cells embedded in the tissue and figure 12b shows a closer look at the embedded tissue.

Figure 12: Left to right; a) A 10X magnification image of a clarified kidney stained with DAPI and imaged under fluorescence microscope clearly highlights the cells embedded within the tissue; b) A 40X magnification image of the same sample gives a closer look at the alignment of cells around glomeruli.

34

Sample 3 – Liver Figure 13a and 13b shows a clarified liver tissue at different magnifications.

Figure 13: Left to right; a) A 20X magnification image of clarified liver stained with fluorescein and imaged under fluorescence microscope shows the arrangement of hepatocytes within the matrix of liver tissue; b) A 40X magnification image of the same sample.

35

Sample 4 – Liver Figure 14a shows a whole clarified liver clearly showing the hepatocytes embedded in hydrogel and 14b shows a higher magnification image of the same sample. Figure 14c&14d are comparisons between H&E of a clarified tissue and normal tissue.

Figure 14: Left to right (clockwise); a) A 4X magnification image of clarified liver stained with fluorescein and imaged under fluorescence microscope shows the arrangement of hepatocytes within the matrix of liver tissue; b) A 40X magnification image of a smaller section of the same sample; c) A control mouse liver H&E sectioning showing striations and highly collagenated tissue with hepatocytes on surface78; d) A clarified liver H&E section showing less or no striations and less collagenated tissue with cells embedded in it allowing to understand the role of CLARITY in high resolution imaging. 36

Decellularization and CLARITY The following sets of images are fluorescent microscopic images of decellularized and clarified samples which show the voids of cellular locations in the organ due to the absence of cell as a result of decellularization. Sample 1 – Kidney Figure 15a showing just the voids in the sample from where the cells have been removed and it outlines the shape of a kidney and 15b shows a closer looks at ECM structure.

Figure 15: Left to right; a) A 4X magnification image of decellularized and clarified kidney highlighting the voids from where the kidney cells have been decellularized; b) A 40X magnification image of the clarified kidney highlights the scaffold interlinking.

37

Sample 2 – Kidney Figure 16 shows the same structure similar to the previous sample.

Figure16: Left to right; a) A 4X magnification image showing voids of cells that are absent and also highlighting the vasculature on the outer surface of the kidney.

38

Sample 3 – Liver Figure 17 shows the outer layer of liver tissue after decellularization and CLARITY were performed on the same sample.

Figure 17: A decellularized and clarified liver scaffold stained with fluorescein shows voids where once cells were present before decellularization. More images and results can be found in the following shared Google Drive folder: https://drive.google.com/open?id=0Bxa725JsDgnTYTZON0JibzJvSnM

39

Decellularization Liver

- 4X -

CLARITY Liver

Kidney

- 10X -

Decellularization+CLARITY Liver Kidney

Kidney

- 20X -

- 4X -

- 10X -

- 4X -

Figure 18: A brief comparison of anatomical and immunohistological results of all the procedures highlighting the difference and comparing the ultra structural definitions of each organs that the individual procedures provide. Left to right; a) A decellularized liver scaffold and fluorescent microscopic image of that scaffold on the column below; b) A decellularized liver scaffold and fluorescent microscopic image of the scaffold; c) A clarified liver after being cleared for 6 weeks, stained with fluorescein (resulting in the the fluorescent yellow appearance of the tissue) and a fluorescent microscopic image of the tissue; d) A clarified kidney after being cleared for 3 weeks and a fluorescent microscopic image of the tissue; e) A liver sample after decellularization and CLARITY performed with 2 weeks clearing time and a fluorescent microscopic images showing the voids of where the cells were present before decellularization; f) A kidney sample after decellularization and CLARITY performed with 2 weeks clearing time and a fluorescent microscopic images showing the voids of where the cells were present before decellularization, 40

5. Discussions/Conclusion

41

5.1.

Decellularization

Decellularization of whole organs is a well known and frequently performed procedure. Nevertheless, there is a lot of details to be investigated using various methods of analysis including mechanical testing, biological assays, and imaging techniques65(Refer to Figures 810). A fluorescent imaging of the liver scaffold clearly highlights the vasculature and scaffold structure which could further be used in 3D cell culturing in synthetic scaffolds to replicate the host tissue’s ultrastructure31. Further to performing decellularization, the standard approach is to recellularize the obtained ECM scaffold with the same type of cells from another host or host’s differentiated stem cells to evidence the capability of the scaffold to regenerate the whole organ and provide vasculature to the cells 66. In the near future, researchers could also use pluripotent stem cells or embryonic stem cells to culture on a decellularized ECM scaffold to analyse the potency of stem cells and the importance of scaffold in the regeneration of whole organs67. This method would be extremely useful in investigating the potency of embryonic stem cells to develop into neuronal, hepatocytic parenchymal and parietal cell types which are still under research. Also in the future decellularization can be done as a regular procedure in tissue engineering organizations to provide patients around the world who are on organ transplant waiting lists with new organs with their cells, reducing the cases of organ rejection and restoring function29,4–6,10,36,37.

Decellularization of the kidney on the other hand although successful, lacks to provide structural details to depth while imaging analyses are performed. Further research can implement different protocols and imaging methods to obtain results with structural precision4–6,10–16,22–25,27–29,31, . Considering the period of time since decellularization has been under practice, one can expect to have a clearer and standard procedure. Further research could be done to provide a database of procedures for different species and organs 38.

What next? Decellularization can be used in future clinically for organ transplantation worldwide. And more research needs to be done on brain decellularization.

42

5.2.

CLARITY

CLARITY on liver and kidney was performed successfully in this project. Both liver and kidney samples provide fine details of the ultrastructure when clarified. One can evidently see the structure of glomeruli in the clarified kidney and also the liver cells aligned in 3dimensional organ scaffolds. Confocal microscopy couldn’t be performed on any clarified sample due to additional costs involved in the procedure. But fluorescence microscopy imaging has provided plenty of details on the whole organ structure thus proving the capabilities of the procedure (Refer to figures 11 -14).

CLARITY is a time-consuming procedure and involves a lot of reagents and materials. In this project, CLARITY was performed keeping the cost in mind and alternatives were used to reduce the costs involved in the procedure. An alternative proposed by researchers in Deisseroth lab to reduce the time taken by the procedure is to use an electrolytic tissue clearing (ETC) chamber which would reduce the time consumed, from few weeks to few days. But the downside of the ETC chamber being the additional costs involved which makes it a tough choice for beginners3,42,71. But to understand the ultrastructure of whole organs, CLARITY seems to be a crucial procedure thus justifying the costs involved.

Currently, CLARITY has been performed over the past five years on mouse brain as a way to mapping the nervous system. This as may sound effective is only a step to the ultimate goal of understanding the neural networks of the human brain which is much more complicated and different to a mouse brain2,44,64,72. Nonetheless, CLARITY seems to be promising enough to achieve the goal of complete understanding of the human neural networks thus contributing not only to the field of biomedical engineering but also opening doors to neuroscience, tissue engineering, virtual rehabilitation, artificial intelligence and much more.

The comparison between a normal H&E and a clarified H&E shows the importance of collagenated scaffold and lipid bilayers in an organ and the absence of them leads to better and clearer analysis of ultrastructures of an organ. Performing H&E on a clarified sample proves the performance of CLARITY and the presence of cells but does not serve any specific purpose.

43

Clarified samples exhibit a phenomenon of swelling and the tissue samples are enlarged in size due to hydrogel polymerization, and this disrupts the structure of vasculature thus making arterial fluid flow analysis hard to achieve. Another aspect of CLARITY to remember while performing it on any organ is that the protocol cannot be standardised for all the organs and species. CLARITY is a highly tissue dependent procedure and requires detailed attention towards the procedure involved. The protocol used in the project suits liver tissues in a higher level than kidney tissues. And also, the fact that CLARITY samples cannot be stored for a very long period of time after clearance makes it a concern. The samples are reported to be forming residual SDS precipitates and the longest period the tissues can be stored for is up to 6 months. In the future, both these concerns can be addressed and thus increasing the reliability of the procedure18,41,43,46–54,56–58,73–76.

And at the end, the debate stands if the procedure is worth the time and the costs involved. For beginners CLARITY may not be suitable both finance and time wise but in the future when CLARITY is more reliable, consistent and standardised, it can serve the purpose for many in the fields of biomechanics, tissue engineering, neurologists, oncologists and much more77.

What next? CLARITY could be used on cancer affected organs to see the effects of cancer cells on the organ and organ scaffold. This could bring a lead in the cancer research field. Also, CLARITY can be used in brain-computer interfacing, artificial intelligence and organ on a chip disciplines to improve understanding of organ functionalities.

5.3.

Decellularization and CLARITY

Combining decellularization and CLARITY has been performed for the first time in this project, and this opens a new interesting topic of discussion, and it also provides an alternative perspective on the structure of an organ as compared to CLARITY. The hydrogel seems to leave the voids empty on the decellularized scaffold where cells were once present, making the voids highly fluorescent when imaging under a fluorescence microscope. This is in contrary to the expectation that performing both decellularization and CLARITY on the same organ might hugely alter the scaffold structure thus leaving the sample structure-less and disintegrated.

44

The size of the decellularized scaffold changes in very small range as compared to just CLARITY performed on the organs. Further research can be done to understand the cause of minimal swelling in a decellularized scaffold when CLARITY is performed.

Combining decellularization and CLARITY set up a platform to compare the results to understand the contrast and 3D dimensional placements of cells in the organ hence leading to understanding the whole 3D orientation of the organ. This knowledge can be used widely in 3D cell culture to replicate the properties of an organ.

What next? Decellularization and CLARITY performed together can again be used in many aspects including tissue engineering, biomaterials, biomechanics, etc.

45

Self Review/ Project Management

The project posted many delays starting from tissue availability and reagents to experiments taking a different path than expected. Sticking to the original plan was as hard as thought it would be but finishing in time was crucial and had to drop important procedures like confocal microscopy imaging. The project had to be cost effective and making and expensive project fit into a budget was a challenge on its own. Nevertheless, it was all a wonderful experience and keeping track of work progress made the project a little less hectic. And since all the steps were planned ahead and before Christmas vacation, even though the project was delayed till April, I managed to finish earlier than expected. Maintaining records will prove to be crucial. Just need to aware of possible delays and outcomes from the beginning.

46

6. Abbreviations

47

DNA – Deoxyribonucleic acid ECM – Extracellular matrix SDS – Sodium dodecyl sulphate TBP – Tributyl phosphate EDTA - Ethylenediaminetetraacetic acid GAG- glycosaminoglycans PBS – Phosphate buffer saline NTIRE – Non-thermal Irreversible Electrporation CLARITY -Clear Lipid-exchanged Anatomically Rigid Imaging/Immunostaining-compatible Tissue hYdrogel SPIM - Selective Plane Illumination Microscopy DAPI – 4’6-diamidino-2-phenylindole H&E –Hematoxylin & Eosin bp* – base pair dsDNA** – double-stranded DNA ng** – nano gram

48

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