optimizing culture medium and protocol for mouse

WAGENINGEN UNIVERSITY AND RESEARCH

BACHELOR THESIS

Optimizing culture medium and protocolfor mouse intestinal stem cell organoid

growth and 2D organoid forming

Author:Steven POOS

Supervisor:Ignacio MIRO ESTRUCH

Laura DE HAAN

Examiner:Dr. Ir. Hans BOUWMEESTER

January 14, 2019

http://www.wur.nl

iii

List of Abbreviations

Caco-2 AdenoCarcinoma of the colon 2ChGA ChromoGranin ACt Threshold CycleDMEM/F12 Dulbecco’s Modified Eagle Medium / F12DMSO DiMethylSulfOxideEGF Epidermal Growth FactorFCS Fetal Calf SerumGAPDH GlycerAldehyde 3-Phosphate DeHydrogenaseHBSS Hanks Balanced Salt SolutionHEPES 4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acidISC Intestinal Stem CellLGR5+ Leucine-rich repeat-containing G-protein coupled Receptor 5 +Lys2 Lysosome 2MIO Mouse-derived Intestinal OrganoidMTT 3-4,5-diMethylThiazol-2-yl-2,5-diphenylTetrazolium bromideMuc2 Mucin 2OGM Organoid Growth MediumP/S Penicillin/StreptomycinPBS Phosphate-Buffered SalineRCF Relative Centrifugal ForceROCK RhO-assoCiated protein KinaseRT-qPCR Reverse-Transcription quantitative Polymerase Chain ReactionSDS Sodium Dodecyl SulfateTA Transit AmplififyingVil1 Villin 1WST-1 Water-Soluble Tetrazolium salt 1

v

Contents

1 Introduction 1

2 Literature search 52.1 Culture medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.2 Organoid growth and passage . . . . . . . . . . . . . . . . . . . . . . . . 92.3 2D organoid forming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102.4 Organoid characterization . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.4.1 RT-qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.4.2 Cell viability and metabolic activity assay . . . . . . . . . . . . . 12

3 Experimental setup 153.1 Culture medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153.2 Organoid and cell line seeding and passage . . . . . . . . . . . . . . . . 17

3.2.1 3D Organoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.2.2 Caco-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.3 2D Organoid Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183.4 RT-qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183.5 Cytotoxicity assay (WST-1 and MTT) . . . . . . . . . . . . . . . . . . . . 19

4 Results 214.1 Culture medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224.2 Organoid passage ratio and time . . . . . . . . . . . . . . . . . . . . . . 244.3 Organoid characterization . . . . . . . . . . . . . . . . . . . . . . . . . . 24

4.3.1 RT-qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244.3.2 Cytotoxicity assay . . . . . . . . . . . . . . . . . . . . . . . . . . 26

5 Discussion 295.1 Culture medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305.2 Organoid passage ratio and time . . . . . . . . . . . . . . . . . . . . . . 315.3 Organoid characterization . . . . . . . . . . . . . . . . . . . . . . . . . . 31

5.3.1 RT-qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315.3.2 Cytotoxicity assay . . . . . . . . . . . . . . . . . . . . . . . . . . 32

5.4 Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Bibliography 35

vi

Appendices AA Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CA.1 Organoids passaging/subculturing . . . . . . . . . . . . . . . . . . . . . CA.2 Dual WST-1-MTT MIO Assay . . . . . . . . . . . . . . . . . . . . . . . . D

A.2.1 2D Organoid seeding procedure . . . . . . . . . . . . . . . . . . DA.2.2 WST-1 incubation procedure . . . . . . . . . . . . . . . . . . . . DA.2.3 Exposure procedure . . . . . . . . . . . . . . . . . . . . . . . . . DA.2.4 MTT assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E

A.3 Caco-2 MTT Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FA.3.1 Caco-2 passaging . . . . . . . . . . . . . . . . . . . . . . . . . . . FA.3.2 Cell seeding procedure . . . . . . . . . . . . . . . . . . . . . . . . FA.3.3 Exposure procedure . . . . . . . . . . . . . . . . . . . . . . . . . FA.3.4 MTT assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G

A.4 RT-qPCR MIO Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HA.4.1 2D Organoid seeding procedure . . . . . . . . . . . . . . . . . . HA.4.2 RNA isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HA.4.3 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . IA.4.4 qPCR and Read-out procedure (SYBR Green) . . . . . . . . . . . IA.4.5 Data analysis, use and management . . . . . . . . . . . . . . . . J

A.5 Primer sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JB DMEM/F12 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KC B27 vs. N2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OD Data management plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q

1

Chapter 1

Introduction

The intestines is a part of the gastrointestinal tract, among others responsible forthe digestion, nutrient absorption and waste elimination (Wells and Melton, 1999).Its surface consists of a highly folded epithelium of villi, microvilli, and intestinalcrypts, dramatically increasing the total surface area to facilitate greater nutrientabsorption. Epithelial cells are of critical importance in maintaining immune home-ostasis within the intestinal tissue, regulating the interactions between the intestinalcells and commensal and pathogenic bacteria (Fair, Colquhoun, and Hannan, 2018).

Intestinal cells have a high turnover rate, completing a full epithelial renewalin approximately 5 days in mice (Barker, Wetering, and Clevers, 2008). This rapidrenewing process is driven by leucine-rich repeat-containing G-protein coupled re-ceptor 5 (LGR5+) intestinal stem cells. LGR5+ stem cells reside in the crypts (Figure1.1), in which they give rise to all different cell types present in the gut epithelium,such as enterocytes, goblet cells, enteroendocrine cells and Paneth cells (Barker et al.,2007). Enterocytes are the most common cell type found in the epithelium, mainlyfunction in nutrient absorption and the protection of the epithelial surface by form-ing an active barrier (Snoeck, Goddeeris, and Cox, 2005). The goblet cells secrete gel-forming mucins, a vital component of the intestinal mucosal layer, which togetherwith resident microbiota forms the first line of defense against toxic compoundsand pathogenic microorganisms (Lievin-Le Moal and Servin, 2006; Ganz, 2002). En-teroendocrine cells form the basis of the largest endocrine system in the body, se-creting multiple regulatory molecules which control physiological and homeostaticfunctions (Rehfeld, 1998; Moran et al., 2008). Paneth cells are positioned in the cryptregion alongside the mulitpotent stem cells, in contrast to the differentiated cell linespreviously mentioned (Figure 1.1). These cells play a crucial role in maintaining thestem-cell niche by producing a large proportion of the small intestinal antimicrobialoutput and by sustaining the proliferative intestinal stem cells by the secretion ofgrowth factors (Richmond and Breault, 2010; Hooper, 2015). These cells are visiblein Figure 1.1.

To understand the interactions that happen in our intestines, researchers havedeveloped many different in vitro- and in vivo models over the years. Traditionalresearch mostly relied on colon cancer cell lines such as Caco-2 (Wang et al., 2017),

2 Chapter 1. Introduction

FIGURE 1.1: Schematic picture of small intestine. Present are therepetitive villus and crypt structures. These structures are composedof LGR+ intestinal stem cells, which can differentiate in Paneth cells,which will stay in the crypt region, and Transit Amplifying (TA) cells,which migrate upwards to the villus region. TA cells divide rapidlyand can differentiate into goblet cells, enteroendocrine cells, and en-

terocytes.

Source: www.stemcell.com

and because of its mutant characteristics, like unrestrained mitosis, these cell linesare efficiently passaged and can grow and differentiate into epithelial monolayers.The downside is that mutative cancer cell lines (like Caco-2) possess non-physiologiccharacteristics, such as differences in tight junction composition and enzyme expres-sion caused by the absence of intestine-specific non-enterocytes, like Paneth cells,goblet cells, enteroendocrine cells and intestinal stem cells (Sun et al., 2008; Pearce etal., 2018). Animals models have been of great value in fundamental research, but itsvalidity is also criticized as a result of the differences in cellular context, physiology,and genetics of different species (Iakobachvili and Peters, 2017). Moreover, animaltesting is seriously reduced the last decades because of ethical considerations. Thischange is triggered by the formulation of the 3Rs (replacement, reduction and refine-ment) in The Principles of Humane Experimental Technique (Russell and Burch, 1959)

To setup a model more representing the nature of intestines, Sato et al createdintestinal organoids derived from single Lgr5+ intestinal stem cells (ISCs). Theseorganoids are self-organized and physiologically-relevant, while also able to growlong-term and stay genomically stable (Kim, Spence, and Takayama, 2017). Grow-ing in a thick medium (Matrigel) which, together with the culture medium, mimics

Chapter 1. Introduction 3

the environment of the intestinal epithelium, stem cells grow and differentiate intocrypt domains on the outside and into villus domains on the inside of the organoid(Figure 1.2). The villus-like epithelium lines off a lumen area in the center of theorganoid, resembling the inner-gut environment (Sato et al., 2009). Moreover, intesti-nal organoids have been shown to exhibit the same functions of intestinal epitheliumoccurring in vivo, including mucus production, epithelial regenerative capacity andabsorptive and secretory functions (Zachos et al., 2016). These functions confirmthat different cell types found in gut epithelium are present in intestine stem cell-derived organoids, including LGR5+ intestinal stem cells, goblet cells, Paneth cells,and enteroendocrine cells (Figure 1.2).

FIGURE 1.2: Schematic view of intestinal organoid. Visible are thedifferent cell types also present in normal gut epithelium: LGR+ cellsand Paneth cells in the crypt domain; enterocytes, goblet cells, and

enteroendocrine cells in the villus domain.

Source: O’Rourke et al., 2016

Although this breakthrough has provided for significant advancements in thein vitro research of gut epithelium, there are several limitations connected to theorganoid culture technology. For instance, the 3D geometry of the organoids pre-vents access to the apical epithelium, hindering studies focused on intestinal inter-actions between epithelium and microorganisms, toxic compounds and food com-ponents. Also, gaining result by usage of conventional microscopy is challengingas Matrigel embedded organoids exist in multiple planes (Wang et al., 2017). Toovercome these limitations, researchers have created a solution that unfolds the 3Dorganoid structure into two-dimensional cell structures (Scott et al., 2016; Moon et

4 Chapter 1. Introduction

al., 2014; VanDussen et al., 2015). Cultures of these intestinal monolayers are ofhigh importance for the development of gut research, not only for its easier accessto the apical epithelium, but also the measurements on interactions between the ep-ithelium layer and microorganisms or different (toxic) compounds can be done in amore standardized way (Hee et al., 2018).

Various protocols have been transcribed to create a confluent monolayer derivedfrom intestinal stem cells, but to make it perform adequately remains a challenge. Byattempting to produce a robust and fully reproductible confluent monolayer, prob-lems with self-renewal (Scott et al., 2016) and characterization (Moon et al., 2014;VanDussen et al., 2015) are leading causes of failure. Because of the high variety ofused media, growth factors and concentrations, the forming of one universal proto-col for producing a proper functioning monolayer seems to be a puzzle to complexto solve.

As mentioned in the paragraph above, researchers have used different proto-cols to form intestinal epithelium organoids, as well as for the creating of planarorganoids. Therefore, this study aims to establish a best practice protocol based onpublished literature for the forming of intestinal organoids and the growing of its2D structure. This study focuses on mouse stem cell-based models, with organoidsderived from mouse adult ISCs, isolated from the ileum.

To form three-dimensional or two-dimensional organoids, the culture mediumplays a big part in the mimicking of the intestinal epithelial environment. Multiplestudies related to this subject use a wide range of media, supplements and growthfactors to form this medium (Wang et al., 2017; Wielen et al., 2016; Thorne et al.,2018). Because of pricey supplements and the differences of concentrations and pro-portions of these supplements in the in papers described culture medium, it is stillnot known how to form a universal medium. Therefore, this study also aims tocreate an optimized and economically viable culture medium based on publishedliterature.

The use of stem cell-derived intestinal epithelium organoids has caused improve-ments in the in vitro gut research field, but this model can be used more efficientlywith an universal protocol. However, this 3D model has the potential to be of greateruse than research, as scientists are experimenting with an approach to use organoidsfor personalized cancer medicine (Aboulkheyr Es et al., 2018). The production of arobust and reproducible culture of two-dimensional monolayers is the next step inunderstanding the mechanisms underlying intestinal epithelium development anddiseases. This model is ideal for the performing of toxicological studies, as the toxiccompounds can be easily added into the accessible lumen area.

5

Chapter 2

Literature search

In this part of the project, extensive research and comparison in literature is done toformulate the materials and methods that are used in the experiments performed inthe lab. Several papers are analyzed to ultimately form an optimized medium forthe growing of 3D and 2D organoids and to describe the best practice protocol forthe forming of these organoids.

2.1 Culture medium

To formulate the content of the optimized culture medium, it is important to firstlydo extensive research to form this medium. The medium used for the growing oforganoids must supplement the intestinal stem cells with vitamins, proteins, growthfactors and other components to optimize cell proliferation and differentiation, whilealso preventing premature apoptosis of cells. This medium simulates the intestinalenvironment to the highest possible degree. Several components and growth factorsare needed to accomplish this aspiration.

In the literature, a large variety of supplements, growth factors and inhibitorsare used. Also, a significant difference exist in the used concentrations of these com-pounds in culture media mentioned in different papers. This makes it difficult toformulate one best practice medium just from literature search. Therefore, in thisstudy, a comparison is made between the culture media used in various papers todefine essential components of the medium. The basal media used in the literatureare displayed in Table 2.1, the used supplements in the culture medium in Table 2.2,and the different growth factors in Table 2.3.

TABLE 2.1: Media used as basal medium in different papers. Theleft column displays the different media, the right column shows the

papers in which the specific media is used.

Components References

DMEM/F12 medium Wielen et al., 2016

Advanced DMEM/F12 medium Wang et al., 2013; Thorne et al., 2018; Kozuka etal., 2017; Liu et al., 2018

6 Chapter 2. Literature search

TABLE 2.2: Supplements used in culture medium described in differ-ent papers. The left column displays the different supplements, themiddle column mentions the different concentration/proportion inwhich the specific components are added to the culture media, andthe right column shows the papers in which the specific components

in specific concentrations/proportions is used.

Components Concentration/Proportion References

N-2 supplement 1x a Thorne et al., 2018; Kozuka et al.,2017; Liu et al., 2018

B-27 supplement 1x Thorne et al., 2018; Kozuka et al.,2017; Liu et al., 2018

Penicillin/Streptomycin 100 µg/ml, 1:1, 1x Wang et al., 2013; Wielen et al., 2016;Thorne et al., 2018; Kozuka et al.,2017; Liu et al., 2018

HEPES 10 mM Wang et al., 2013; Thorne et al., 2018;Kozuka et al., 2017

GlutaMAX 1x Wang et al., 2013; Wielen et al., 2016;Thorne et al., 2018; Kozuka et al.,2017; Liu et al., 2018

N-acetyl-cysteine 1 mM Wang et al., 2013; Thorne et al., 2018;Kozuka et al., 2017; Liu et al., 2018

aAt some occasions, a proportion of “1x” is given. This annotation means that the supplement isconcentrated one time in the medium. Normally, these supplements are bought as hundred timesconcentrated (100x). Thus, “1x” signifies a hundred times dilution of the supplied substance.

The most significant part of the medium consist of a 1:1 mix of Dulbecco Modi-fied Eagle Medium and Ham’s F12 Medium called DMEM/F12. This medium mix,containing vitamins, amino acids and salts, supports the growth of rodent cells invitro adequately (Yao and Asayama, 2017).

Many papers use advanced DMEM/F12 over ‘regular’ DMEM/F12 medium (Ta-ble 2.1). Advanced DMEM/F12 contains several proteins (e.g., Albumax II), reduc-ing agents (e.g., glutathione), trace elements ( e.g., sodium selenite) and other com-ponents (e.g., ethanolamine, sodium pyruvate) that are not present in DMEM/F12medium (Appendix B).

However, advanced DMEM/F12 does not contain L-glutamine which ‘regular’DMEM/F12 does. Therefore, advanced DMEM/F12 medium is often enriched withGlutaMAX-I. GlutaMAX-I is a dipeptide, L-alanine-L-glutamine, which is more sta-ble than L-glutamine in aqueous solutions and does not degrade spontaneously(Tritsch and Moore, 1962). Cells cultured with GlutaMAX-I gradually release amino-peptidases which hydrolyze the dipeptide, slowly releasing the monopeptides L-alanine and L-glutamine in the culture media. These peptides are then taken up bythe cells and used for protein production or in the TCA cycle. The result is an ef-ficient energy metabolism and a higher growth yield (Hassell, Gleave, and Butler,1991). This makes GlutaMAX-I an improved cell culture supplement that can beused as a direct substitute for L-glutamine.

2.1. Culture medium 7

The supplements B27 and N2 are added to the medium in the literature, as canbe seen in Table 2.2. B27 is a serum-free supplement normally used to support thegrowth of embryonic neuron cells. N2 is developed for in vitro differentiation ofmouse embryonic stem cells. All components present in N2 can also be found inB27, although in unknown concentrations. Additionally, several components in N2and B27 are present in advanced DMEM/F12 medium, though in different concen-trations (Appendix C).

The literature makes sure that buffer HEPES, mucus loosener N-acetylcysteineand antibiotics penicillin/streptomycin (P/S) is added to the medium (Table 2.2).

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES in short, is a buffer-ing agent commonly used in cell cultures, as it is better at maintaining pH thanbicarbonate buffers. Also, HEPES is an effective buffering agent for maintainingenzyme structure and function at a low temperature, because, like water, HEPES’dissociation decreases when the temperature lowers (Baicu and Taylor, 2002).

N-acetylcysteine is an antioxidant and mucolytic agent, profoundly used in thegrowth medium for mouse intestinal stem cells. This antioxidant is added becauseof its ability to (partially) inhibit cell apoptosis (Hockenbery et al., 1993).

Penicillin/streptomycin is an antibiotics mixture widely used in mammal cellculture medium to prevent contamination. It is the most common antibiotic solutionin cell culture and it has no harmful effects on the cells themselves (Martínez-Liarte,Solano, and Lozano, 1995).

Many papers make use of the standard growth factor composition ENR (i.e., EGF,Noggin, R-spondin (Table 2.3)). These growth factors are essential for the prolifera-tion and differentiation of intestinal Lgr5+ stem cells.

Epidermal growth factor (EGF) is a protein that stimulates cell proliferation,growth, and differentiation by binding to its receptor, i.e. epidermal growth factorreceptor (EGFR) (Harris, Chung, and Coffey, 2003).

Noggin is a signaling molecule that is involved in the development of many bodytissues, like nerve tissue and muscles (Marcelino et al., 2001).

R-spondin is a protein which plays an important role as a positive regulator ofthe WNT signaling pathway (Jin and Yoon, 2012).

Wnt3a is another important growth factor, which is i.a., produced by Panethcells. Wnt3a is secreted by the Wnt3a gene, one that plays a crucial role in both cellproliferation and differentiation processes in intestinal stem cells (He et al., 2015).Thorne et al., 2018 showed that adding Wnt3a to the medium is not necessarilyneeded to form a monolayer, as Paneth cells already secrete this substance. Theaddition of its counterpart in the growth medium, BMP, is also not needed, as differ-entiated enterocytes produce this growth factor in the villus cells.

Y-27632 is a selective inhibitor of Rho-associated protein kinase (ROCK), an en-zyme that modulates actin cytoskeletal organization and cell contraction by binding


TABLE 2.3: Growth factors used in the culture medium in differentpapers. The left column displays the different growth factors, themiddle column mentions the different concentrations in which thespecific components are added to the culture media, and the right col-umn shows the papers in which the specific growth factors in specific

concentrations is used.

Components Concentration References

EGF 50 ng/ml Wang et al., 2017; Thorneet al., 2018; Liu et al., 2018;Kozuka et al., 2017

Noggin71 ng/ml Wang et al., 2017

50 ng/ml Thorne et al., 2018

100 ng/ml Liu et al., 2018; Kozuka et al.,2017

R-spondin

75 ng/ml Wang et al., 2017

500 ng/ml Liu et al., 2018; Kozuka et al.,2017

1 µg/ml Thorne et al., 2018

homebrew b Wielen et al., 2016

Y-2763210 µM Wang et al., 2017; Thorne et

al., 2018; Liu et al., 2018

20 µM Kozuka et al., 2017

Wnt-3A



100 ng/ml Kozuka et al., 2017

homebrew Wielen et al., 2016

LDN-193189100 nM Thorne et al., 2018

500 nM Liu et al., 2018

CHIR-990212,5 µM Liu et al., 2018

10 µM Thorne et al., 2018

A-8301 500 nM Wang et al., 2017

Thiazovivin 2,5 µM Kozuka et al., 2017

BMP-4 300 ng/ml Kozuka et al., 2017

Blebbistatin 10 µM Liu et al., 2018

bSome growth factors can be grown at home, as can be seen in the concentrations column of R-spondin and Wnt-3A. This is done because of the relative high price of purchase of these factors. Withthis method, it is very hard to determine what the concentration of these growth factors is in the finalmedium.

2.2. Organoid growth and passage 9

to ATP (Riento and Ridley, 2003). Y-27632 interferes with this process by binding tothe catalytic site of ROCK before ATP does (Davies et al., 2000; Ishizaki et al., 2000).Various studies include ROCK-inhibitor in the culture medium because of its provenenhancement of stem cell survival after passaging by preventing dissociation-inducedapoptosis (Watanabe et al., 2007). More specifically, Y-27632 improves survival ofISC-derived monolayers at the initiation of differentiation protocols (Rezania et al.,2011).

Other inhibitors, like TGFβ-inhibitor LDN-193189, GSK3-inhibitor CHIR-99021,myosin IIA inhibitor Blebbistatin, and ALK5-inhibitor A-8301, are also used in theliterature to strengthen the organoid or monolayer structure, although less oftenthan Y-27632. These inhibitors are mostly used simultaneous with ROCK-inhibitor.In one study (Liu et al., 2018), Blebbistatin is used as a substitute for Y-27632, becauseof its allegedly improved attachment and growth of ISCs (Zhao et al., 2015). Theseinhibitors are of lesser importance than Y-26732 as they are rarely added to culturemedia used in the literature (Figure 2.3).

2.2 Organoid growth and passage

Different studies use different passage times, ratios and techniques. The differencesbetween several papers are displayed in Table 2.4.

TABLE 2.4: Characteristics of growing 3D organoids. The left columndisplays the different characteristics of 3D organoid growth, the mid-dle column mentions the differences in these specific characteristics,and the right column shows the papers in which the specific charac-

teristic is used.

Organoid Growth References

Passage time4 days Wang et al., 2017

7 days Wielen et al., 2016; Kozukaet al., 2017

Passage ratio

1:3 Wang et al., 2017

1:4 Wielen et al., 2016

1:6 Kozuka et al., 2017

1:8 Kozuka et al., 2017

Medium changedEvery 24 hours Wang et al., 2017

Only after passaging Wielen et al., 2016; Kozukaet al., 2017

GelMatrigel Wang et al., 2017; Wielen et

al., 2016; Thorne et al., 2018;Kozuka et al., 2017; Liu et al.,2018

Like earlier mentioned, the epithelium of murine small intestine renews every 5days (Barker, Wetering, and Clevers, 2008). Therefore it seems logical that murineISC-derived organoids are also passaged after approximately 5 days. However, the


literature indicates different passage times, from 4 to 7 days (Table 2.4). A passagetime greater than 5 days can still be adequate as these 5 days is the life expectancy ofonly the differentiated cells in the villus region of the intestinal epithelium (Barkeret al., 2007; Clevers and Bevins, 2013). The organoids will not die after an epitheliumrenewal. Instead, the dead differentiated cells are released from the epithelial layerand form a growing amount of debris in the organoid’s lumen, which is visible inorganoids with dark, necrotic cores (O’Rourke et al., 2016).

The studies displayed in Table 2.4 also give multiple options for the ratio of pas-saging organoid structures, from 1:3 to 1:8. This ratio is correlated to the number ofcrypts present in the to be passaged well. Several studies change the medium duringculturing. The importance of changing the medium is tested in this study.

As can be seen in Table 2.4, all of the researched literature uses Matrigel (Corning)for the forming of organoids. Matrigel is a gelatinous protein mixture and resemblesthe extracellular environment found in many tissues. Because of this resemblance,Matrigel is often used by cell biologists and biochemists as basement membranematrix for the culturing of cells (Benton et al., 2009; Hughes, Postovit, and Lajoie,2010).

2.3 2D organoid forming

2D organoids or monolayers are certainly not formed in the same way in every study.Literature uses different types of gels, plates, equipment, and chemicals for the 2Dformation. The differences are mentioned in Table 2.5.

TABLE 2.5: Characteristics of 2D organoid/monolayer form-ing. The left column displays the different characteristics of 2Dorganoid/monolayer growth, the middle column mentions the dif-ferences in these specific characteristics, and the right column shows

the papers in which the specific characteristic is used.

Monolayer forming References

Gel

Matrigel Wielen et al., 2016; Thorne etal., 2018; Kozuka et al., 2017;Liu et al., 2018

Collagen hydrogel Wang et al., 2017

Plate

6-well plate Wang et al., 2017

24-well plate Wielen et al., 2016

24-transwell plate Kozuka et al., 2017

48-well plate Liu et al., 2018

96-well plate Thorne et al., 2018

For the forming of a confluent monolayer out of murine intestinal ISC-derivedorganoids, the choice for Matrigel as coating of the well surface is a popular one(Table 2.5). Thorne et al., 2018 claims that Matrigel-coated surfaces best supported

2.4. Organoid characterization 11

crypt survival compared with poly-L-lysine or collagen-coated surfaces, while Wanget al., 2017 argues that culture of primary epithelium on Matrigel surfaces produceda short-lived, non-proliferative monolayer of cells.

Various plates with different well surfaces are mentioned in the literature (Table2.5). Also, a Transwell plate is used to grow the monolayer, particularly to determineion transport (Kozuka et al., 2017). The transwell is frequently used in monolayerforming as it provides independent access to both apical and basolateral membranesof the monolayer, but a large amount of viable cells are needed to create a monolayer.

2.4 Organoid characterization

Investigation to classify the optimized culture medium is not done with only a lookthrough the microscope. A microscopic view can say something about the organoid’smorphology, but not about the cell composition within the organoid or its viability.Therefore, organoids can be characterized by a RT-qPCR procedure, as mentionedin the literature (Wielen et al., 2016; Hee et al., 2018; Ettayebi et al., 2016). To acquireinformation about the organoid’s cell viability, assays are described in the literaturewith different reagents, like MTT (Grabinger et al., 2014; Han et al., 2017) and WST-1(Nam et al., 2018).

2.4.1 RT-qPCR

Reverse-Transcription quantitative (real-time) Polymerase Chain Reaction (RT-qPCR)is an analysis method frequently used in the literature for determining the levels oftranscription of specific genes, indicating the presence of different intestinal epithe-lium cells in organoids (Wielen et al., 2016; Hee et al., 2018; Bartfeld, 2016).

To acquire expression levels of indicator genes of intestinal epithelium cells dur-ing RT-qPCR, RNA is first isolated from 2D plated intestinal organoids after 72 hoursof culturing in culture medium, and later transformed to cDNA by reverse transcrip-tion. The cDNA is then amplified by the use of gene-specific primers, and subse-quently detected by the use of dye SYBR Green I. This nucleic acid stain binds tounfolded DNA caused by raised temperatures, and emits green light (Zipper et al.,2004). The amplified DNA sequences are far more abundant than non-amplifiedDNA sequences, making the fluoresce measurements directly related to the amountof the cDNA of interest. These measurements are used to determine the gene ex-pression of the gene that indicates the presence of a specific cell of interest.

The value used for the expression measurements is called the threshold cycle(Ct). This Ct value describes the cycle at which the observed expression first ex-ceeds a fixed threshold expression value (McCall et al., 2014). During the exponen-tial phase of amplification, the amount of each target gene approximately doublesbetween subsequent cycles. This results in a lower Ct value indicating for a higher


gene expression as more gene-specific cDNA exceeds the threshold expression early,while a higher Ct value indicates for a low or no gene expression, as it takes longer toamplify a smaller amount of gene-specific cDNA. Ct values are still reliable enoughfor gene expression indicating at a maximum of 40 (Goni, Garcia, and Foissac, 2009;Mar et al., 2009)

The Ct value is used to calculate the fold change via the delta delta method(Schmittgen and Livak, 2008; Pfaffl, 2001). Equation 2.1 shows the final form ofthis method. This equation is used to compare the gene expression in two differentsamples (sample A and sample B); each sample is related to an internal control gene.The equations 2.2 and 2.3 expand the delta delta method to its full form. Sample Ais used as control sample gene in which gene expression is compared to the samplegene (sample B).

Fold change = 2−∆∆CT (2.1)

Fold change = 2[∆CTsample−∆CTcontrol] (2.2)

Fold change = 2[(CTsampleA−CTsampleB)−(CTcontrolA−CTcontrolB)] (2.3)

The genes of our interest are the indicator genes for the different intestinal ep-ithelium cells: Lgr5+ stem cells, enterocytes, goblet cells, enteroendocrine cells andPaneth cells. To achieve accurate and reliable gene expression results, the expressionof a target gene has to be normalized with a housekeeping gene, which displays con-stitutive expression in all tissues of living organisms during various phases of devel-opment and under different environmental conditions. (Jain et al., 2006; Eisenbergand Levanon, 2003).

2.4.2 Cell viability and metabolic activity assay

MTT (3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) is a non-solubletetrazole used for assessing cell metabolic activity (Stockert et al., 2018). MTT onitself is yellow, but it gets reduced in the mitochondria of living cells to a purple for-mazan (Figure 2.1A). MTT is because of its net positive charge not able to transportthrough the cell membrane, which makes an extra dilution step with dimethylsul-foxide (DMSO) necessary to bring in the results.

WST-1 (water-soluble tetrazolium salt) is a second generation tetrazolium char-acterized by a net negative charge and therefore largely cell-impermeable (Figure2.1B). In addition, second-generation tetrazoliums are reduced to water-soluble for-mazans at the cell surface, making an assay with this reagent measurable withoutthe necessity to dissolve the cells in DMSO possible (Berridge, Herst, and Tan, 2005).This makes it possible to perform a dual WST-1-MTT assay to test the cells viabilityand metabolic activity. A spectrophotometer measures the activity of both WST-1and MTT.


(A) Yellow MTT to a purple formazan.

Source: Rogan Grant

(B) Pale yellow WST-1 to a dark yellow formazan.

Source: G-Biosciences

FIGURE 2.1: The reduction reactions of both MTT and WST-1 to aformazan

15

Chapter 3

Experimental setup

3.1 Culture medium

As can be seen in the previous chapter, a wide range of supplements, growth fac-tors and inhibitors are used in the literature in different concentrations. We use thisinformation to formulate a total of four different media, and compare them witheach other and with Intesticult Organoid Growth Medium (OGM) (Stemcell Tech-nologies, Cologne, Germany). The medium from Intesticult is used as the standardmedium for the growing of organoids in the Toxicology lab. Therefore, Intesticult’sOGM is used as control medium and compared to the newly formulated media.

A total of four culture media are constructed during this project. The names ofthese media, SP I to SP IV, are based on the writer’s initials (Steven POOS). In thisstudy, SP I and SP II (Table 3.1) are used for the growing of 3D ISC organoids, whileSP III and SP IV (3.2) are used for the forming of 2D organoids.

All culture media contain Advanced DMEM/F12 (Gibco, Waltham, Massachusetts,USA), N2, B27, P/S, GlutaMAX, EGF (Invitrogen, Breda, Netherlands), R-spondin1 and Wnt3a. The latter two growth factors are produced by 293T cells (Cultrex,Gaithersburg, Maryland, USA) and L cells (ATCC, Manassas, Virginia, USA) respec-tively.

TABLE 3.1: Components of the two formulated culture media SP Iand SP II. These media are used for the growing of 3D organoids.

SP I SP II

Advanced DMEM/F12 Advanced DMEM/F12N-2 supplement (1x) N-2 supplement (1x)B-27 supplement (1x) B-27 supplement (1x)penicillin/streptomycin (1:1, 1x) penicillin/streptomycin (1:1, 1x)GlutaMAX (1x) GlutaMAX (1x)EGF (50 ng/mL) EGF (50 ng/mL)R-spondin 1 (3x diluted) R-spondin 1 (6x diluted)Wnt3a (30x diluted) Wnt3a (30x diluted)

16 Chapter 3. Experimental setup

TABLE 3.2: Components of the two formulated culture media SP IIIand SP IV. These media are used for the growing of 2D organoids.

SP III SP IV

Advanced DMEM/F12 Advanced DMEM/F12N-2 supplement (1x) N-2 supplement (1x)B-27 supplement (1x) B-27 supplement (1x)penicillin/streptomycin (1:1, 1x) penicillin/streptomycin (1:1, 1x)HEPES (10 mM) HEPES (10 mM)GlutaMAX (1x) GlutaMAX (1x)N-acetylcysteine (1mM) N-acetylcysteine (1mM)EGF (50 ng/mL) EGF (50 ng/mL)Noggin (100 ng/mL) Noggin (100 ng/mL)R-spondin 1 (2,5x diluted) R-spondin 1 (2,5x diluted)Wnt3a (25x diluted) Wnt3a (25x diluted)Y-27632 (10 µM)

The difference between SP I and SP II, visible in Table 3.1, is the proportion ofWnt3a : R-spondin in the culture medium, 1:10 in SP I and 1:5 in SP II. These pro-portions are based on the proportions used in Thorne et al., 2018 and Kozuka et al.,2017 respectively. Both R-spondin and Wnt3a are not bought in known concentra-tions but homebrewed, which makes it difficult to determine the concentrations ofthese growth factors in the culture medium. That is why dilution rates are given.

The components of the two 2D culture media SP III and SP IV are shown inTable 3.2. These media also contain HEPES (VWR International, Poole, England), N-acetylcysteine (Janssen Chimica NV, Beerse, Belgium) and Noggin (R&D systems,Minneapolis, Minnesota, USA). The composition of SP III differs with SP IV be-cause of the addition of Y-27632 (Selleckchem, Munich, Germany) to the medium.R-spondin and Wnt3a are added in a different dilution compared to SP I and SP II.

The 3D culture media SP I and SP II are used in a comparison assay. In this assay,three different 24-wells plates are filled with 4/5 droplets of 50 µL crypt-containingMatrigel and solidified after incubation in humidified conditions. OGM, SP I andSP II are added to the different plates as culture medium (Figure 3.1). The crypts aregrown into organoids and after 6/7 days replated in a 1:2 ratio into new well plates.This is done for a total of three passages. Microscopic investigation is performed ev-ery day (except during weekends) and the amount of organoids per well are writtendown.

The 2D culture media SP III and SP IV are used for the RT-qPCR assay, explainedin Section 3.4.

3.2. Organoid and cell line seeding and passage 17

(A) (B)

(C)

FIGURE 3.1: Schematic view of culture medium assay setup. For thisassay OGM (A), SP I (B) and SP II (C) are used as culture medium.

3.2 Organoid and cell line seeding and passage

3.2.1 3D Organoids

Organoids are grown in droplets of 50 µL Matrigel (Corning, Amsterdam, Nether-lands) in 24 wells plates (Corning, Amsterdam, Netherlands), with 1 mL of culturemedium added (OGM, SP I, or SP II). The organoids are passaged by a protocol stan-dardized by the Toxicology department. This protocol, displayed in Appendix A.1,describes the dissociation and replating of crypts in new wells, forming organoid-like structures in approximately 5 days.

The protocol for organoid passage is used to try out the differences in passageratio mentioned in the literature. This is done by inserting two dissociated wellsinto one conical tube. After centrifugation, the supernatant of approximately 2 mLinstead of 1 mL is removed. Another way is by changing the amount of Matrigelused to homogenize the crypt containing pellet after the centrifugation step from150 µL to 100 µL or 50 µL, replating the organoids in ratios of 1:3 or 1:2 respectively.

A refreshment of culture medium is also tested. In this examination the mediumis refreshed 24 hours after passaging. The differences in passage ratio and the re-freshing of culture medium are both tested with OGM as culture medium.

3.2.2 Caco-2

Caco-2 cells are grown in T75 tissue culture flasks (Corning, Amsterdam, Nether-lands) with DMEM (Gibco, Waltham, Massachusetts, USA), 10% FCS, 1% P/S and1% non-essential amino acids (Invitrogen, Breda, Netherlands). These cells grow onthe surface of the tissue culture flasks, and are passaged when 50-70% confluence isreached. The cells are first detached from the flask surface by the use of a Trypsin

18 Chapter 3. Experimental setup

solution (Gibco, Waltham, Massachusetts, USA), and replated in a new culture flaskin a ratio of 1:4 to 1:6. The full protocol of the culturing and passaging of Caco-2cells is displayed in Appendix A.3. This cell line is used for comparison during thecytotoxicity assay described in Section 3.5.

3.3 2D Organoid Growth

In this study, intestinal organoids are grown in 2D for the performing of two dif-ferent assays, the cytotoxicity assay with WST-1 and MTT, and the RT-qPCR assayfor the organoid characterization. 96 wells plates (Corning, Amsterdam, Nether-lands) are used for the cytotoxicity assay and 48 wells plates (Corning, Amsterdam,Netherlands) are utilized for RT-qPCR. For one well on a 96- or 48 wells plate, fourwells from a 24 wells plate full of 3D organoids are used for the 2D culture. Theorganoids are dissociated and centrifuged, as is described in Appendix A.1. Afterthe supernatant is removed, the crypt pellet is resuspended with assay medium,which is culture medium (OGM, SP III or SP IV) supplemented with 10% Fetal CalfSerum (FCS) (Bodinco BV, Alkmaar, Netherlands) . The crypts are developed into2D organoids after 3 days of incubation under humidified conditions, 37oC and 5%CO2. After this, the 2D culture is ready to be used for the above mentioned assays.

3.4 RT-qPCR

Gene expression levels is measured with an RT-qPCR assay. Organoids are grown in2D with SP III and SP IV as used culture media, described in Section 3.3. After 3 daysof culturing, RNA is isolated from the organoids by the use of spin columns, collec-tion tubes, buffers and reagents supplied from the QIAshredder- and RNeasy minikit (Qiagen, Venlo, Netherlands). This RNA is than reverse transcripted to cDNA byusage of Quintitect Reverse Transcription kit (Qiagen, Venlo, Netherlands). Primers(Qiagen, Venlo, Netherlands) are used to determine the presence of intestinal stemcells, enterocytes, goblet cells, enteroendocrine cells and Paneth cells in the two di-mensional MIOs by amplifying the cDNA sequences of marker genes. GAPDH isused as housekeeping gene (Qiagen, Venlo, Netherlands). The genetic codes of thedifferent forward and reverse primers are displayed in Table A.3, obtained fromWielen et al., 2016. SYBR Green I (Qiagen, Venlo, Netherlands) is used to stain theamplified cDNA sequences. The complete protocol for this assay can be found inAppendix A.4. As explained in Section 2.4.1, the obtained Ct values are used tocalculate the fold change. The fold change values display the gene expression ofa sample gene (sample B) relative to a chosen control sample gene (sample A), thehousekeeping gene (GAPDH) is used as internal control.

3.5. Cytotoxicity assay (WST-1 and MTT) 19

3.5 Cytotoxicity assay (WST-1 and MTT)

The cell viability of the organoids in 2D is examined by the use of WST-1 (Sigma,Osterode am Harz, Germany) and MTT (Sigma, Osterode am Harz, Germany). Afterthree days of culturing, WST-1 reagent is added to the 2D culture and incubated forthree hours at humidified conditions, after which the absorbance of the medium ismeasured via a spectrophotometer (Molecular Devices, San Jose, California, USA).WST-1 is used to define the initial viable cell density per well before MTT exposure.These results are used to correct the eventual MTT results after exposure. The 2Dplated organoids are grown in OGM.

For comparison, the human colon cancer cell line Caco-2 is plated in a 96-wellsplate and incubated for 24 hours at humidified conditions, after which it can be usedfor the MTT assay.

TABLE 3.3: Different concentrations of Triton X-100 added to cell cul-tures. Assay medium of the Caco-2 culture is the same as the cul-ture medium, as its culture medium already exist of 10% FCS. MIO =

Mouse-derived Intestinal Organoids.

Cell cultureTriton X-100

Stock conc. (%) Intermediate conc. (%) Final conc. (%)in PBS in assay medium in assay medium

MIO

1 0.02 0.010.5 0.01 0.0050.1 0.002 0.0010 0 0

Caco-2

1 0.02 0.010.5 0.01 0.0050.1 0.002 0.0010 0 0

Next, different concentrations of Triton X-100 (Sigma, Osterode am Harz, Ger-many) is added to the medium (Table 3.3) and exposed to the cells for 24 hours inhumidified conditions, before MTT is added. Triton X is a known cytotoxic agent,and therefore used as positive control. MTT needs two hours of exposure, before thecells are lysed by DMSO (Acros, Geel, Belgium) and the amount of formed formazanproduct is measured in a spectrophotometer.

The protocol for the WST-1 MTT assay of mouse-derived intestinal organoids(MIO) can be found in Appendix A.2, the MTT assay of Caco-2 cells is visible inAppendix A.3. This protocol is composed by Ignacio MIRO ESTRUCH.

21

Chapter 4

Results

Organoids are grown from crypts of intestinal stem cells. After organoids are dis-sociated during passaging, the crypts are replated in new Matrigel and with newculture medium. These crypts are visible as little round circles (Figure 4.1A). After24 hours, the proliferative cells form rapidly a spheroid-like structure, and apop-totic cells already fill the newly formed lumen, visible as the darker middle partof the structure (Figure 4.1B). Another 24 hours later the first sights of buddingtakes place in parallel form, visible in Figure 4.1C. You can also see here that thespheroid keeps increasing in size and the lumen turns darker by addition of moreapoptotic cells. After 72 hours, budding has taken place in multiple planes of thenow formed organoid, creating the characteristic organoid structure. Figure 4.1Dshows the organoid with some parts out of focus, indicating the structure to be 3-dimensional.

FIGURE 4.1: The transition of crypt to an organoid. Pictures are takenafter 0 (A), 24 (B), 48 (C), and 72(D) hours of incubation. Organoids

are formed in every tested medium in this study.

22 Chapter 4. Results

4.1 Culture medium

The formulated culture media SP I and SP II are compared to one another and to theOGM medium in an assay described in Chapter 3. The amount of budding structuresin a well are counted by looking through the microscope, and an average of visibleorganoids per well of a plate is calculated. These averages make up the bar lengthsin Figure 4.2.

FIGURE 4.2: Organoids grown in OGM, SP I and SP II as culture me-dia.The day of culturing and the different passages are given on theX axis. The amount of organoid found per well is situated on the Yaxis. The bar height of a specific bar is based on the average organoidnumber per well, incubated on a plate with a specific growth medium

after specific days of culturing in a specific passage.

Several things can be said about the performance of the different growth mediain Figure 4.2. The control medium, OGM, increases in amount of organoids afterseveral passages. SP I has the highest number of organoids per well in the firstpassage. In the second passage, the organoid number of SP I is comparable to SPII, but both decrease in organoid density after 7 days. The third passage displaysno data about SP II after one day of culturing. This is caused by contamination ofseveral wells in SP I and SP II after 7 days in passage 2 and the contamination of thewhole SP II plate in passage 3.

We observed that mature organoids are formed from crypts grown in both cul-ture media SP I and SP II, as well as in the control medium OGM (Figure 4.3). Nextto these budding structures, hollow structures known as spheroids were also foundin the three culture media (Figure 4.4).

4.1. Culture medium 23

(A) OGM

(B) SP I

(C) SP II

FIGURE 4.3: Organoids grown in OGM (A), SP I (B), and SP II (C)after 7 days of culturing.

FIGURE 4.4: Spheroids found in OGM, SP I and SP II respectively.

Regarding the organoid : spheroid ratio in the organoid cultures, differences areobserved between the three culture media. For OGM culture, we observed a 1:2ratio for the first days of culturing, while after 7 days this ratio is flipped to 2:1.For SP I and SP II culture, we see more budding structures in earlier stages, whicheventually results into an organoid : spheroid ratio of 5:1 and 6:1 for SP I and SP IIcultures respectively.

Considering the above mentioned results, we can conclude that the formulatedmedia SP I and SP II performs similarly to control medium OGM, while contribut-ing to a better organoid culture in terms of organoid : spheroid ratios. Differences


between the two constructed media SP I and SP II seem neglectable.

4.2 Organoid passage ratio and time

As mentioned in Chapter 3, the protocol for passaging MIOs (Appendix A.1) is usedto experiment with the passage ratio and passage time. We see that the effectivenessof a used passage ratio is strongly correlated to the organoid density of the to bepassaged well. For a well grown in OGM with 50+organoids, the passage ratios 1:5and 1:6 deliver wells with a poor organoid concentration, while ratios of 1:4 and 1:3mostly give wells with a rich amount of organoids. For wells grown in OGM with30-50 organoids, a passage ratio of 1:4 delivers a poorly amount of organoids, 1:3gives a moderate organoid concentration per well, and 1:2 renders wells rich withorganoids. Wells with a organoid number lower than 30 are not capable of deliveringwells with a high organoid density, and are therefore only used for passaging whenthey are combined with wells with a higher organoid concentration.

We see a declining of organoids after three passages using a passage time of 5days, while an increase of organoids per well is observed with a passage time of7 days, when grown in OGM. In addition, successful passages are observed withpassage times up to 11 days.

The medium change after 24 hours does not make a noticeable difference in 3Dorganoid growth. Organoids are formed with the same rate when the OGM mediumis not changed.

After experimenting with several variables in the 3D organoid forming protocol,we can say that, given a well with approximately 60 organoids grown in OGM, it isready to be passaged after 7 days and is best to passage in a 1:3 ratio. This culturemedium used in this well does not need to be changed after 24 hours.


4.3.1 RT-qPCR

TABLE 4.1: Ct values of four examined genes amplified by RT-qPCRin 2D MIOs grown in SP III and SP IV. The tested cDNA is diluted 64

times to make expression measurement possible.

GenesCt value

SP III SP IV

ChGA 30.79 29.86Vil1 26.17 24.78Lyz2 35.01 33.88GAPDH 22.72 24.4


(A)

(B)

FIGURE 4.5: Gene expression of the three tested cell-specific genesVil1, ChGA and Lys2. (A) The log 2 fold change of Vil1 is comparedto the log 2 fold change of ChGA and Lys2 for organoids grown in SPIII and SP IV. Vil1 is not displayed as it is used as control sample genein this comparison, making its log 2 fold change values equal to 0. (B)The fold change of Vil1, ChGA and Lys2 expressed in SP III and SPIV are compared with each other. All Ct values of SP III are used ascontrol sample gene is this comparison, making its fold change values

equal to 1.


The presence of intestinal epithelial cells in 2D plated MIOs grown in SP III andSP IV is tested in a RT-qPCR assay. Gene expression levels are measured in Ct-values, which are used to calculate the fold change values via the delta delta method,as is described in Section 2.4.1. The log 2 fold change between Vil1 and ChGA andbetween Vil1 and Lys for SP III and SP IV are visible in Figure 4.5A. The Vil1 val-ues are not given, as they are used as control sample gene (sample A) in the deltadelta equation. This makes the fold change values of Vil1 equal to 1, and its Log 2value equal to 0 in this figure. The fold change values of Vil1, ChGA and Lys2 in2D organoids grown in SP III relative to the same genes expressed in 2D organoidsgrown in SP IV is displayed in 4.5B. Here, all Ct values of SP III are used as controlsamples in comparison to SP IV, making all their fold change values equal to 1. Thespecific Ct values of the cell-indicating genes can be found in Table 4.1.

The log 2 fold change values given in Figure 4.5A show comparable results be-tween the expression of the genes ChGA and Lys2 compared to Vil1 in SP III and SPIV. This indicates that Paneth cells and enteroendocrine cells are present in the sameproposition to enterocytes present in organoids grown in SP III and SP IV.

We see that Lys2 has higher negative values than ChGA. This means that the geneindicating for Paneth cells is expressed less than the indicator gene for enteroen-docrine cells. Furthermore, the enteroendocrine cell-indicator is less expressed thanthe indicator gene for enterocytes. These results indicate that there are more ente-rocytes than enteroendocrine cells en Paneth cells present in organoids grown in SPIII and SP IV, while more enteroendocrine cells are present in these organoids thanPaneth cells are.

Figure 4.5B displayes a difference in gene expression of the tested cell-specificgenes between SP III and SP IV. We see that SP III shows less expression of the in-dicator genes Vil1, ChGA and Lys2 when comparing to SP IV. This indicates thepresence of more differentiated cells in organoids grown in SP IV than in organoidsgrown in SP III.

4.3.2 Cytotoxicity assay

The cell viability of two-dimensionally plated MIOs are tested by exposure to dif-ferent concentration of known cytotoxic agent Triton X. The results are received bythe MTT cell death treatment and compared to MTT assay results of Caco-2 cellsexposed to the same concentrations of Triton X.

Section 3.5 describes the use of the WST-1 incubation step before the MTT expo-sure assay, namely to define the initial vial cell density per well before different con-centrations of Triton X is added. The utility of this extra defining step is displayed inFigure 4.6. We see that the unnormalized results show a linear like declining of ab-sorbance with increasing Triton X percentages, while the normalized results show a


FIGURE 4.6: Normalized and unnormalized absorbance levels inpercentages of 2D MIOs incubated with different concentrations ofTriton-X at 450 nm minus the background absorbance at 620 nm. Thevial cell density of each well was defined with an WST-1 exposureassay before the MTT cell death treatment in the normalized results.

(A) (B)

FIGURE 4.7: Cytotoxicity measurements of 2D plated MIOs com-pared to Caco-2 cells at different concentrations of Triton X. The ab-sorbance levels of the 2D MIO culture after exposing with MTT arenormalized with WST-1. (A) The absorbance of both cell types at dif-ferent Triton X concentrations are compared to one another. (B) Theabsorbance levels of 2D MIO and Caco-2 are given in percentages.This is done by translating the absorbance of 2D MIOs and Caco-2

cells with no added Triton X to 100%.


more expected plateau like decline of absorbance between 0.001% and 0.005% of Tri-ton X. Thus, the use of an WST-1 assay before the MTT exposure assay is an effectiveway to normalize the differences in cell density of 2D plated MIO culture.

In Figure 4.7, we compare the viability of the Caco-2 cells and the 2D MIOs intwo different ways: by absorbance (Figure 4.7A), and by percentage (Figure 4.7B).We see that at non-lethal concentrations of Triton X and with no added Triton X,0.001% and 0% respectively, the absorbance of 2D plated organoids is considerablylower than the Caco-2 cells’ absorbance (Figure 4.7A). At a lethal concentration ofadded Triton X (0.01%), both cell lines display comparable low absorbance. Whenthe absorbance values of the 2D MIOs and Caco-2 cells without any Triton X addedare rephrased as 100%, we see comparable results between the two cell lines (Figure4.7B). This means that both 2D MIOs as Caco-2 cells react in a similar matter to anincreasing concentration of Triton X. Thus, the viability of 2D MIOs is on par withthat of Caco-2 cells.

29

Chapter 5

Discussion

Mouse derived intestinal organoids have been used in research for nearly a decadesince its first formation (Sato et al., 2009). The forming of 2D organoids and organoid-derived monolayers are already known subjects in papers, even though its first de-scription was just four years ago (Moon et al., 2014). Difficulties that come with theformation of organoids in vitro are particularly based on the composition of the com-plex culture medium, which, together with Matrigel as extracellular matrix, mimicsthe environment of the intestinal epithelium to such a degree that ISCs can grow anddifferentiate into an artificial intestine structure, called an organoid. Research seeksfor the forming of a best practice culture medium, but because of its complexity, ahuge variety of supplements, growth factors and inhibitors are part of different cul-ture media described in literature. To make it even more complicated, these culturemedium components are used in different concentrations and proportions in differ-ent studies. Additionally, protocols used in research to form 3D or 2D organoidsdiffer from each other in a fundamental way.

In this study, we constructed a total of four culture media based on culture mediaused in the literature for the growing of 3D and 2D organoids. We used the first twoculture media, SP I and SP II, for the 3D culturing and compared them with a controlmedium, OGM. We also tested the protocol for culturing organoids standardizedby the Toxicology department by using different passage ratios and -times and bychanging the medium, all based on previously described protocols in the literature.Next to this, we investigated the presence of intestinal epithelial cells in 2D platedorganoids grown in SP III and SP IV to examine the influence of ROCK-inhibitorY-27632 to a 2D culture. Finally, we examined the organoid’s cell viability via anewly designed dual WST-1-MTT assay and compared the results with the MTTassay results of colon cancer cell line Caco-2.

In this chapter, we discuss the results that we found and compare them withresults displayed in the literature. Conclusions are drawn about the construction ofthe culture medium and the formulation of a protocol for 3D and 2D organoids, andrecommendations for future studies are made in Section 5.4.

30 Chapter 5. Discussion

5.1 Culture medium

The culture media described in Chapter 3 are used for different assays, SP I and SPII for 3D growth and SP III and SP IV for 2D growth, respectively. Initially, it wasplanned to produce three different media, which would differ from each other bythe presence or absence of Y-27632 and Wnt3a. The formulation of four differentculture media is caused by the prolonged delivery time for Noggin and Y-27632,which resulted in the altered formulation of the first two culture media, SP I andSP II. HEPES and N-acetylcysteine were initially part of the formulation of SP I andSP II, but were left out by mistake. SP III and SP IV were formed during a laterstage, when Y-27632 and Noggin were available, but time shortage caused these twoculture media to only be applied by the forming of 2D MIOs in the RT-qPCR assay.

For the growth of mouse ISC-derived organoids in 3D, the constructed culturemedia SP I and SP II seem to perform in a decent matter. In Figure 4.2 it is visiblethat more crypts were grown into budding structures in these constructed mediathan in the control medium, OGM, for the first two passages. However, the amountof organoids per well does not say everything about the quality of a medium, asspheroids are also formed during culturing (Figure 4.4). It is still unclear if thesespheroids are able to grown into organoids after they are dissociated and replated.Some papers claim that spheroids do not transit into organoids, even after contin-uous passaging (Mustata et al., 2013; Fordham et al., 2013), while others argue thatspheroids can transit into organoids within the same passage (Navis et al., 2018).

The culture medium test showed that the formulated culture media SP I and SPII performed better than control medium OGM, as more crypts grown in SP I orSP II eventually became mature organoids compared to crypts grown in OGM. Thedetected organoid : spheroid ratio of 2:1 for OGM and 5:1 and 6:1 for SP I and SP IIcultures respectively visualizes the differences clearly.

Differences between the culture media SP I and SP II seem to be neglectable. InFigure 4.2, both culture media follow a comparable path in the second passage, andsimilar budding structures are formed in both media. Also, the organoid : spheroidratio of both culture media are comparable. As said in Section 3.1, the R-spondin :Wnt3a propositions stated in Thorne et al., 2018 and Kozuka et al., 2017 were usedin the composition of the culture media SP I and SP II respectively. Thus, the use ofthese different propositions in culture media cause organoids to grow in a similarmatter.

The media SP I and SP II are, due to mistakes and circumstances out of our con-trol, not in the exact way formulated as we initially wanted. HEPES, N-acetylcysteineand Noggin where the components that would be added initially, but were eventu-ally left out. Sato et al., 2009 showed that, when an organoid culture is grown in amedium with no added Noggin, the amount of organoids visible per well decreases

5.2. Organoid passage ratio and time 31

after every passage, to eventually a very small number is reached in passage three.In this study we see that the amount of organoids per well increases after in passagetwo when grown in culture media without Noggin, in contrast with Sato et al., 2009.This can be caused by the a considerable percentage of Paneth cells which producedNoggin to such a degree that the organoids can survive the passages.

5.2 Organoid passage ratio and time

Section 4.2 shows that a passaging ratio of 1:4 is only usable when a well with 50+organoids is passaged, while wells with a organoid number between 30 and 50 re-quire a 1:3 or 1:2 passage ratio. This data is comparable to the passage ratios givenin Wang et al., 2017 and Wielen et al., 2016, as stated in Table 2.4. This shows thatthe ideal passage ratio of an organoid-containing well is highly influenced by itsorganoid concentration. Thus, it is advisable to count the amount of organoids in awell before passaging to determine its ideal passage ratio.

The best practice passage time used in this study is 7 days. This is in agreementwith Wielen et al., 2016 and Kozuka et al., 2017, also stated in Table 2.4. Passagetimes can however be increased to utmost 11 days, which does not agree with theliterature mentioned in Table 2.4, but does with the passage times used in Kasagi etal., 2018 and Young et al., 2018. However, the passage time used in this study is notonly based on previously mentioned times in the literature. As stated in O’Rourkeet al., 2016, organoids need to be passaged when they show a dark, necrotic cores,while the crypt regions are still visible. The organoid is not viable anymore whenthe whole structure has turned dark and when no distinctive budding structurescan be seen. Taking everything in consideration, we advise to passage a organoidculture grown in OGM every 7 days, or when the organoids show dark cores whilestill possessing viable budding regions.


5.3.1 RT-qPCR

Figure 4.5A indicates that more enterocytes are present in organoids grown in SP IIIand SP IV than enteroendocrine cells en Paneth cells. This is an expected result, asthe villus region takes up the most room on the intestinal epithelium and the largestpart of this villus consists of enterocytes (Snoeck, Goddeeris, and Cox, 2005). Theseresults are also in agreement with the Vil1 expression in the 2D culture of VanDussenet al., 2015. Next to that, we see that more enteroendocrine cells are present in the2D plated organoids grown in SP III and SP IV than Paneth cells are. Not muchcan be found related to this proportion in the literature. However, it is known thatenteroendocrine cells make up approximately 1% of the epithelial cell population(Sternini, Anselmi, and Rozengurt, 2008).

32 Chapter 5. Discussion

In figure 4.5B is visible that genes indicating for differentiated cells are expressedmore in SP IV than in SP III. This indicates that 2D organoids grown in a culturemedium with ROCK-inhibitor Y-27632 contain less differentiated cells than organoidsgrown in a culture medium without Y-27632. These are surprising results, as ROCK-inhibitor enhances stem cell survival by preventing dissociation-induced apoptosis(Watanabe et al., 2007), and also promotes mouse embryonic stem cell differentia-tion (Kamishibahara, Kawaguchi, and Shimizu, 2014; Kamishibahara, Kawaguchi,and Shimizu, 2016).

Due to circumstances out of our control we were not able to use the primersMuc2 and Lgr5. This declares our incomplete results in the Rt-qPCR assay. Also,due to mistakes and time shortage, no calibration curves are used for the calculationof the results. This is why the results are presented in relative numbers.

5.3.2 Cytotoxicity assay

The results of the dual WST-1-MTT assay performed on both MIOs and Caco-2 cellsshow that the 2D plated intestinal organoids react to the toxic compound Triton X ina similar matter as Caco-2 cells (Figure 4.7B). This is in contrast with Grabinger et al.,2014, in which is concluded that intestinal organoids and Caco-2 cells differentiallyrespond to various cell death triggers. However, this paper does not describe theresponse of organoids and Caco-2 cells to known cytotoxic agent Triton X.

Although the 2D MIOs and Caco-2 cells display comparable relative results todifferent concentrations of Triton X, the results show a big difference in absorbancebetween the two cell lines (Figure 4.7A). This can be caused by the amount of cellspresent in the wells of the tested cell lines. As a cancer cell line, Caco-2 grows into amonolayer after 24 hours of culturing. Although intestinal organoids have 72 hoursof culturing before the assay, it was still not possible for the MIOs to grow in a con-fluent monolayer. The crypts formed 2D budding structures, which do not fill upthe well surface completely. However, other factors can also have its influence onthe absorbance levels. Factors like differences in cell density, mitochondrial activity,metabolic activity or initial viability are all plausible causes.

5.4 Recommendations

It is recommended to re-perform the organoid growth assay for collecting more datafrom more passages. The results of a new assay can support the results described inthis study to provide for full closure, also regarding the importance of Noggin in theculture medium.

The conclusions about the passage ratios and -times in this study are based on 3Dgrown organoids in OGM. This can be different with organoids grown in differentculture media, like SP I, SP II, SP III or SP IV. Therefore it is advisable to re-examine

5.4. Recommendations 33

the ideal passage ratio and -times when a different culture medium is used in futureresearch.

The RT-qPCR is also recommended to redo, as the surprising results regardingthe influence of ROCK inhibitor Y-27632 on the development of differentiated celllineages ask for further research. Also, next to Muc2 and Lgr5, other primers canbe used next to the intestinal epithelial cell indicators, i.e., primers indicating forproliferative cells. This assay can also be expanded by testing the gene expression oforganoids grown in OGM, SP I and SP II next to SP III and SP IV, or by examiningthe gene expression of organoids grown in 3D compared to organoids grown in 2D.Moreover, the gene expression of Caco-2 cells can be compared to the MIOs geneexpression by RT-qPCR. In addition to the RT-qPCR assay, another characterizationexperiment, immunostaining, can be used for the characterization of organoids.

The newly designed dual WST-1-MTT assay could also be used to determine thedifferences in cell viability of 2D MIOs grown in the four constructed media SP I,SP II, SP III and SP IV, and the control medium OGM. For further investigations,we recommend to perform this assay to determine the differences between the cul-ture media more in depth. Additionally, the viability of 2D plated organoids can becompared to the 3D plated organoid’s viability in this assay. This expansion of theWST-1-MTT assay is also recommended to test the difference in viability betweenthe 3D and 2D culture. Lastly, other lethal exposures can be used, for instance TNFαor X-ray irradiation.

As stated in Section 3.1 as well as in Appendix A.1, 50 µL of Matrigel is used perwell. This is based on an earlier used protocol of the Toxicology, and based on theproduct description paper delivered with Intesticult’s OGM (Stemcell Technologies,2016). After all experimenting was done, an older paper, previously overlooked,was found claiming that 50 µL of Matrigel is abundant, as a volume of 20 µL alsomeets the requirements (Jeude et al., 2015). Therefore, is is recommended to testthe differences in the volume of Matrigel used for organoid passaging in furtherresearch.

35

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Nam, Myeong-Ok et al. (2018). “Effects of a small molecule R-spondin-1 substituteRS-246204 on a mouse intestinal organoid culture”. In: Oncotarget 9.5, pp. 6356–6368. ISSN: 1949-2553. DOI: 10.18632/oncotarget.23721. URL: http://www.oncotarget.com/fulltext/23721.

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O’Rourke, Kevin P et al. (2016). “Isolation, Culture, and Maintenance of Mouse In-testinal Stem Cells.” In: Bio-protocol 6.4. ISSN: 2331-8325. URL: http : / / www .ncbi.nlm.nih.gov/pubmed/27570799http://www.pubmedcentral.nih.gov/

articlerender.fcgi?artid=PMC4996657.




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http://dx.doi.org/10.1093/bioinformatics/btu239

http://dx.doi.org/10.1093/bioinformatics/btu239



http://dx.doi.org/10.1038/mi.2013.98

http://www.nature.com/articles/mi201398

http://dx.doi.org/10.1177/1756283X08093943




http://dx.doi.org/10.1016/J.CELREP.2013.09.005



http://dx.doi.org/10.18632/oncotarget.23721

http://www.oncotarget.com/fulltext/23721

http://www.oncotarget.com/fulltext/23721

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C

Appendix A

Protocols

A.1 Organoids passaging/subculturing

After 5-7 days, the organoids have formed a significant amount of debris in its lu-men, resulting in a very dark structure. At this moment, the crypts need to be pas-saged to start the organoid forming procedure all over again. Crypts are passagedin a 1:4 or 1:3 wells ratio in duplo.

First, the organoid growth medium (OGM) is removed from the well with a 1mL pipette. The polarized Matrigel droplet is now clearly visible. Then, the Ma-trigel is dissociated by scratching the surface of the well and pipetting up and downwith 1 mL DMEM/F12 (Gibco, Waltham, Massachusetts, USA). Air bubbles willform, which is not an issue. 1 mL of the Matrigel debris, disrupted organoids, andDMEM/F12 substance is inserted into a 10 mL conical tube (Corning, Amsterdam,Netherlands).

The conical tubes are spun off in a centrifuge (Sigma, Osterode am Harz, Ger-many) for 10 min , 750 RCF. The supernatant is discarded and the crypts containingpellet is resolved in 150 ul Matrigel. The substance is homogenized by pipetting upand down several times. Thereafter, 50 µl of the Matrigel is added to the center of 4different wells on a pre-heated 24-wells plate.

The plate is then incubated for 10-15 min at 37 ◦C, 5% CO2 (Thermo Scientific,Waltham, Massachusetts, USA) to polymerize the Matrigel. After this, 1 mL of OGMis added to each well via the side wall to avoid contact with the Matrigel. SterilePBS (Gibco, Waltham, Massachusetts, USA) is added to every unused well to re-duce evaporation of medium. The plate is placed in the incubator again, and neworganoids are formed in approximately 5 days.

D Appendix A. Protocols

A.2 Dual WST-1-MTT MIO Assay

A.2.1 2D Organoid seeding procedure

First, prepare 2x diluted Matrigel (in organoid medium) and add 35 ul to each wellof a 96-well plate.

Incubate the plate for 15-20 minutes at 37oC and 5% CO2 to make the matrigel so-lidify.

Take the plate containing mature 3D MIOs (multiple buds) from the incubator, dis-card 2/3 of the medium from every well and mechanically dissociate the organoidsto obtain the corresponding crypts.

Transfer the crypt suspension into a 10 mL tube and centrifuge at 1000 RCF for 5minutes at 4oC.

Remove the supernatant and re-suspend the crypt pellet with organoid mediumsupplemented with 10% FCS.

Count via microscopic evaluation the crypt density (number of crypts per mL).

Seed the intestinal crypts in the 48-well plate containing 2x Matrigel pre-coated wellswith a density of around 20 crypts per well. To do so use 150 ul of crypt suspensionper well.

Let crypts develop into 2D organoids for 72 hours by incubating the plate underhumidified conditions, 37oC and 5% CO2. During this time, microscopic evaluationof the organoids is carried out to check the attachment and correct growth of the 2Dorganoids.

A.2.2 WST-1 incubation procedure

Add 15 ul of WST-1 reagent solution (10% v/v) to all the wells (150 ul). Incubate theplate for 3 hours at humidified conditions, 37oC and 5% CO2.

Take supernatant medium (75 ul) and measure the absorbance at 450 nm (FormazanDye) and 620 (background signal) using a spectrophotometer.

Create an excel file which will contain both raw and processed data.

Subtract the background (Abs = 620 nm) from the formazan signal (Abs = 450 nm).

Express the results relative to the medium so that you acquire a normalization factor.

A.2.3 Exposure procedure

In this assay, exposure to Triton-X-100 is carried out on day 3. To do so, the first stepis the preparation of the respective Triton-X-100 suspensions in dispersant/solventand culture medium (hereafter also called exposure medium). The main steps aredepicted in Table A.1.

A.2. Dual WST-1-MTT MIO Assay E

Prepare stock (in PBS) and intermediate (assay medium) concentrations using tubes,flat 24-well plates or U-shaped 96-well plates.

Once prepared, add 75 ul of the intermediate medium (2x conc.) to the alreadyexisting 75 ul (from seeding) in each well of the flat 96-well plate (containing 2Dorganoids) to reach the final concentrations of Triton-X-100 (Table A.1).

TABLE A.1: Different concentrations of Triton X-100 added to MIOcell cultures.



MIO

1 0.02 0.010.5 0.01 0.0050.1 0.002 0.0010 0 0

Next, incubate the plate for 24 hours at humidified conditions, 37oC and 5% CO2.

A.2.4 MTT assay

Add 15 ul of MTT reagent solution (10% v/v) to all the wells 2 hours before the endof the 24h exposure.

Incubate the plate for these last 2 hours of exposure at humidified conditions, 37oCand 5% CO2.

Discard the medium, add 20 ul of 2% SDS and incubate for 60 minutes.

Add 130 ul of DMSO to lyse the cells and dissolve the formazan product.

Measure the absorbance at 562 nm (Formazan Dye) and 620 (background signal)using a spectrophotometer.

Create an excel file which will contain both raw and processed data.


Correct the data from each well by their Normalization factor.

F Appendix A. Protocols

A.3 Caco-2 MTT Assay

A.3.1 Caco-2 passaging

When reaching 50-70% confluence, cells are passaged in a ratio of 1:4 to 1:6.

Remove medium and wash the cells with 10 mL PBS.

After washing, incubate with 1.5 mL Trypsin solution for 5 minutes under humidi-fied conditions, 37oC and 5% CO2.

Add 8.5 mL of culture medium (it neutralizes trypsin) and after mixing, add therequired volume of cell suspension and new culture medium (different dependingon the desired ratio) in a new T75 cell culture flask and incubate under humidifiedconditions, 37oC and 5% CO2 until reaching 50-70% confluence again.

A.3.2 Cell seeding procedure

When Caco-2 cells reach a confluence of 50-70% of the tissue culture flask, dissociate(trypsinize) the cells as described in the previous section.

After adding the 8.5 mL of culture medium, mix thoroughly to obtain a single cellsuspension and transfer 20 ul to an Eppendorf tube.

Add 20 ul of Trypan Blue solution (Sigma, Osterode am Harz, Germany) to the ex-isting 20 ul of cell suspension and pipette 20 ul of this mix into a cell counter slide.

Count/Obtain the viable cell concentration (C1) present in the initial cell suspen-sion (S1) using Cellometer AutoT4 (Promega, Leiden, Netherlands) equipment andsoftware.

Calculate (C1*V1=C2*V2) the volumes of the initial cell suspension (V1) and culturemedium required to prepare a new cell suspension (S2) of 2 x 10 5 cells/mL.

Add 100 ul of the newly made Caco-2 cell suspension (S2) in each well of a flat-transparent 96-well plate what results in a cell density of 2 x 10 4 cells per well.

Let cells attach for 24 hours under humidified conditions, 37oC and 5% CO2.

Next, microscopic evaluation of the cells is performed to check for possible growth-or attachment defaults or systematic cell seeding errors.

A.3.3 Exposure procedure

In this assay, exposure to Triton-X-100 is carried out on day 3. To do so, the first stepis the preparation of the respective Triton-X-100 suspensions in dispersant/solventand culture medium (hereafter also called exposure medium). The main steps aredepicted in Table A.2.

Prepare stock (in PBS) and intermediate (in organoid medium + 10% FCS) concen-trations using tubes, flat 24-well plates or U-shaped 96-well plates.

A.3. Caco-2 MTT Assay G

Once prepared, add 75 ul of the intermediate medium (2x conc.) to the alreadyexisting 75 ul (from seeding) in each well of the flat 96-well plate (containing 2Dorganoids) to reach the final concentrations of Triton-X-100 (Table A.2).

TABLE A.2: Different concentrations of Triton X-100 added to Caco-2cell cultures.



Caco-2

1 0.02 0.010.5 0.01 0.0050.1 0.002 0.0010 0 0

Next, incubate the plate for 24 hours at humidified conditions, 37oC and 5% CO2.

A.3.4 MTT assay

Add 20 ul of MTT reagent solution (10% v/v) to all the wells 3 hours before the endof the 24h exposure.

Incubate the plate for these last 2 hours of exposure at humidified conditions, 37oCand 5% CO2.

Discard the medium, add 150 ul of DMSO to lyse the cells and dissolve the formazanproduct.

Measure the absorbance at 562 nm (Formazan Dye) and 620 (background signal)using a spectrophotometer.

Create an excel file which will contain both raw and processed data:


H Appendix A. Protocols

A.4 RT-qPCR MIO Assay

A.4.1 2D Organoid seeding procedure

First prepare 2x diluted Matrigel (in organoid medium) and add 100 ul to each wellof a 48-well plate.

Incubate the plate for 15-20 minutes at 37oC and 5% CO2 to make the matrigel so-lidify.

Take the plate containing mature 3D MIOs (multiple buds) from the incubator, dis-card 2/3 of the medium from every well and mechanically dissociate the organoidsto obtain the corresponding crypts.

Transfer the crypt suspension into a 10 mL tube and centrifuge at 1000 RCF for 5minutes at 4oC.

Remove the supernatant and re-suspend the crypt pellet with organoid mediumsupplemented with 10% FCS.

Count via microscopic evaluation the crypt density (number of crypts per mL).

Seed the intestinal crypts in the 48-well plate containing 2x Matrigel pre-coated wellswith a density of around 40 crypts per well. To do so use 400 ul of crypt suspensionper well.

Let crypts develop into 2D organoids for 72 hours by incubating the plate underhumidified conditions, 37oC and 5% CO2. During this time, microscopic evaluationof the organoids is carried out to check the attachment and correct growth of the 2Dorganoids.

A.4.2 RNA isolation

Add 300 ul of RLT Lysis Buffer (Qiagen), break mechanically the Matrigel structureand shake the plate.

Transfer the lysate to a QIAshredder spin-column tube and centrifuge (Eppendorf,Nijmegen, Netherlands) at 8000 RCF (30 seconds) in order to homogenize and filterout insoluble debris.

Add 300 ul of Ethanol 70% (1:1 Dilution) to the already filtered lysates.

Put the 600 ul of sample/ethanol mix in an RNeasy spin-column tube and centrifugeat 8000 RCF for 30 seconds.

Discard content of the collection tube and reuse.

Add 700 ul of RW1 buffer (Qiagen) and centrifuge at 8000 RCF (30 seconds).


Add 500 ul of RPE buffer (Qiagen) and centrifuge at 8000 RCF (30 seconds).


A.4. RT-qPCR MIO Assay I

Add again 500 ul of RPE buffer (Qiagen) and centrifuge at 8000 RCF (30 seconds).

Put spin-columns without adding anything in new collection tubes and centrifugeat 8000 RCF for 1 minute (to completely dry off the filter).

Place spin-columns in new Eppendorf tubes and add 30 ul of RNAse free water andcentrifuge at 8000 RCF for 1 minute.

Put the RNA samples in ice or store them at -20oC to use later on.

A.4.3 Reverse Transcription

Measure the amount (ng/ul) and quality (Abs260/280) of the RNA by using a Nan-odrop technology (Isogen, De Meern, Netherlands). To do so, after setting the blank(only water), add 1 ul of each sample and annotate the respective results.

Calculate the amount of sample required to transform 300 ng of RNA into cDNA(Vol:12 ul).

Add 2 ul of gDNA WipeOut (Qiagen) buffer to the 12 ul of sample containing 300ng RNA and incubate at 42 oC for 2 minutes.

Next add to each sample 4 ul of Quantiscript RT buffer (Qiagen), 1 ul of QuantiscriptReverse Transcriptase (Qiagen) and 1 ul of Quantiscript RT primer mix (Qiagen) andincubate for 15 minutes at 42 oC followed by 3 minutes at 95 oC in a thermal cycler(Bio-RAD, Hercules, California, USA).

Put the cDNA samples in ice again or store them at -20oC to use later on.

A.4.4 qPCR and Read-out procedure (SYBR Green)

Prepare primer stock solutions for GAPDH by adding 1.1 mL TE buffer (MolecularProbes, Eugene, Oregon, USA) in the vials containing the lyophilized primers andmaking aliquots of 100 ul. Store the aliquots at -20oC.

Make Standard Curve (SC) dilution series to assess the PCR process. To do so, pool1 ul from each sample and make dilutions in water (4x, 16x, 64x, 256x, 1024x).

Dilute every cDNA sample 64x times (e.g. 1 ul cDNA sample + 63 ul water).

Prepare separate primer mixes containing the following amounts per each sample:

• 12.5 ul of 2x concentrated SYBR Green.

• 2.5 ul Primer.

Add the following volumes to the RotorGene PCR tubes (Qiagen, Venlo, Nether-lands) (total = 25 ul per tube):

• 10 ul of SC sample, NTC, NRTC or 64x diluted cDNA sample.

• 15 ul of GAPDH Mix (depending on the case).

Put all lids, transfer the RotorGene PCR tubes to the rotor and place the lock ring.

J Appendix A. Protocols

Start a run (3-step with melt) of 40 cycles. Each cycle consists in 10 secs at 95 oCfollowed by 15 secs at 60oC and 20 secs at 72oC.

A.4.5 Data analysis, use and management

Using the RotorGeneQ Series Sofware quality parameters such as the results ob-tained for the NTC, NRTC, SC R2, SC efficiency or melting curves (see next sectionfor criteria followed). If quality is satisfactory, continue with the next steps.

Determine all Ct values for every sample.

Create an excel file which will contain both raw and processed data:Criteria and quality check measures used in this assay

Only exposure conditions which led to a viability percentage higher than 80% whencomparing to the medium/vehicle control were used for this assay.

Exposure media has to be freshly prepared immediately before use in the assays.

Microscopical evaluation is constantly carried out to monitor the state of the 2Dorganoids.

Only RNA samples with the Abs260/280 value within 1.8-2,1 were accepted for RT-qPCR.

Melting curve should show no signs of primer dimers or products of different lengths.

A.5 Primer sequences

TABLE A.3: Primers used in RT-qPCR analysis to determine differentepithelial cells (Wielen et al., 2016)

Name primer Used to determine Forward primer Reverse primer

Lgr5 Intestinal stem cellCCTACTCGAA GCATTGGGG

GACTTACCCAGT TGAATGATAGCA

Vil1 EnterocytesTACCTCAAGAC AAAGCCCTTG

CCACCCTGGAA AAGGCAGGGTAG

Muc2 Goblet cellsAGGGCTCGGAA CCAGGGAATCG

CTCCAGAAA GTAGACATCG

ChgA Enteroendocrine cellsCAGGGACACTA GGTGATTGGGT

TGGAGAAGAGA ATTGGTGGCT

Lys2 Paneth cellsATGGAATGGCT ACCAGTATCGG

GGCTACTATGG CTATTGATCTGA

GAPDH Housekeeping geneAGGTCGGTGTG WTGTAGACCATG

AACGGATTTG TAGTTGAGGTCA

K

Appendix B

DMEM/F12 Medium

LA

ppendixB.

DM

EM/F12

MediumFIGURE B.1: DMEM/F12 vs. Advanced DMEM/F12 Medium.

Appendix

B.D

MEM

/F12M

ediumM

FIGURE B.2: DMEM/F12 vs. Advanced DMEM/F12 Medium.

O

Appendix C

B27 vs. N2

PA

ppendixC

.B27

vs.N2

FIGURE C.1: B27 vs. N2 Supplement.

Q

Appendix D

Data management plan

Organizational contextName: Steven POOS

Supervisors: Ignacio MIRO ESTRUCH , Laura DE HAAN

and Dr. Ir. Hans BOUWMEESTER

Short description of your researchThis study aims to formulate an optimized culture medium for the growing of 3Das well as 2D intestinal organoids with information took from the literature. Fur-thermore, the protocol for 3D organoid forming is optimized in this study by testingdifferent variables in the protocol, also based on the literature. In addition, this studyexamines the cell viability of intestinal organoids grown in 3D via a newly designedWST-1-MTT assay, and the results are compared with MTT assay results of Caco-2cells.

Data management rules (who is responsible for what?)I, Steven Poos, am responsible for the storage of the data generated within the thesis.The data management tables provided on Blackboard are used to name the raw datafiles and linked to a more detailed description of the experiments in my lab journal.This is done for all the raw data files that are generated during the thesis. For pro-cessed data files another data management table provided by Blackboard is used. Inthis table, a general description of what the process file is about is given. Also statedis from which raw data file the processed data was obtained. The lab journal will behanded over to the supervisor at the end of the thesis. All the data is stored on W:

\PROJECTS\TOX_Research-data\HansBouwmeester\2018Ignacio\2018StevenPoos andon my personal onedrive account as a backup and to have the possibility to accessthe data from home.

What type of research data is produced?

1. Cytotoxicity data from MTT and dual WST-1-MTT assay. Raw data is pro-duced in the form of MTT absorbance measurements. Processed data is pro-duced in the form of graphs made in Excel.

R Appendix D. Data management plan

2. RT-qPCR is used to study the cell composition of 2D grown organoids. Raw isproduced in the form of Ct values from the RotorGene software output. Pro-cessed data is produced in the form of tables and graphs in Excel

3. A lab journal is kept to record the experiments throughout the project.

Software choices

1. Raw data: RotorGene and spectrophotometer software

2. Processed data: Excel

What is the amount of the data, and how will the amount increase in timeCirca 50 MB, the amount of data will increase over time.

Sharing and ownershipThe data is shared among personnel and students who have access to the ProjectsDrive.W:\PROJECTS\TOX_Research-data\HansBouwmeester\2018Ignacio\2018StevenPoos

Documentation and data management tableThe data management tables provided on Blackboard are used to name the rawdata files and linked to a more detailed description of the experiments in my labjournal. This is done for all the raw data files that are generated during the the-sis. For processed data files another data management table provided by Black-board is used. In this table, a general description of what the process file is aboutis given. Also, it will state from which raw data file the processed data was ob-tained. The lab journal is handed over to the supervisor at the end of the the-sis. All the data is stored on W:\PROJECTS\TOX_Research-data\HansBouwmeester\

2018Ignacio\2018StevenPoos and on my personal onedrive account as a backupand to have the possibility to access the data from home.

Short-term storageShort-term storage is found on the personal WUR account (M-drive).

Long-term storageAs previously mentioned, long-term storage of the data is stored on the server of thewur. The location of the data is W:\PROJECTS\TOX_Research-data\HansBouwmeester\2018Ignacio\2018StevenPoos. An extra backup copy is stored on my personalonedrive account for access of the data at home.

Data management tables

Name Steven PoosRegistration number 97042966400Start date of thesis 1 September 2018Supervisor 1 Ignacio Miro EstruchSupervisor 2 Laura de HaanFile path W:\PROJECTS\TOX_Research-data\Hans Bouwmeester\2018Ignacio\2018 Steven Poos

File name Device Description Processed Y/N

20181026_R_poos003_1 spectrophotometer WST-1 assay 2D MIO, grown in OGM Y20181027_R_poos003_1 spectrophotometer MTT assay 2D MIO, grown in OGM Y20181108_R_poos003_1 spectrophotometer MTT assay Caco-2 cells Y20181121_R_poos003_1 PCR PCR data 2D MIO grown in SP III and SP IV Y

Data management tables

Name Steven PoosRegistration number 97042966400Start date of thesis 1 September 2018Supervisor 1 Ignacio Miro EstruchSupervisor 2 Laura de HaanFile path W:\PROJECTS\TOX_Research-data\Hans Bouwmeester\2018Ignacio\2018 Steven Poos

File name* Program Based on raw file(s) Description Presented in Figure

20181026_P_poos003_1 Excel 20181026_R_poos003_120181027_R_poos003_1

Cytotoxic assay 2D MIO, WST-1 and MTT, grown in OGM, exposed by Triton X

4.6 and 4.7

20181108_P_poos003_1 Excel 20181108_R_poos003_1 Cytotoxic assay Caco-2 cells, MTT, exposed by Triton X

4.7

20181221_P_poos003_1 Excel 20181121_R_poos003_1 PCR data from 2D MIO grown in SP III and SP IV 4.5

optimizing culture medium and protocol for mouse

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