Organoid Culture
Handbook
Matrices | Cells | Reagents
TABLE OF CONTENTS
Introduction to Organoid Culture .....................................................................................
3
Organoid Models................................................................................................................
Featured Products for Organoid Culture ...........................................................................
Organoid Progenitor Cells..........................................................................................
4
5
5
Mouse Small Intestine Organoid Progenitor Cells...........................................
Extracellular Matrices................................................................................................
BME (Basement Membrane Extract) for Organoid Culture.............................
Collagen I .........................................................................................................
Reagents....................................................................................................................
5
6
6
7
7
R-spondin (RSPO1) Expressing Cell Line..........................................................
Proteins and Antibodies .................................................................................
7
8
R-spondin (RSPO1) Purified Proteins .......................................................
8
Wnt (Wnt3A) Proteins ..............................................................................
8
Lgr5 (Gastric Stem Cell Marker) Antibodies.............................................
9
Organoid Harvesting Solution ........................................................................
9
Organoid Culture Protocols ..............................................................................................
General Submerged Method for Organoid Culture ................................................
Crypt organoid culture techniques..........................................................................
Air Liquid interface (ALI) Method for Organoid Culture ........................................
Clonal Organoids from lgr5+ cells ............................................................................
Organoid Culture Examples...............................................................................................
Liver Organoid Culture.............................................................................................
Human Liver.....................................................................................................
Hepatocellular Carcinoma (HCC) ....................................................................
Mouse Liver......................................................................................................
Gastrointestinal Organoid Culture...........................................................................
Small Intestine Organoids................................................................................
Human Colorectal ............................................................................................
Mouse Adenoma .............................................................................................
Mouse Intestinal- Colorectal Cancer Model ...................................................
Transgenic Mouse ............................................................................................
Esophageal Organoid Culture ...................................................................................
Esophageal Cancer ..........................................................................................
Esophageal Barrett’s Epithelial ........................................................................
Esophageal Normal .........................................................................................
Breast Organoid Culture...........................................................................................
Prostate Organoid Culture .......................................................................................
Metastatic Prostate Cancer-Derived Organoids...............................................
Harvested Organoids .......................................................................................
Organoids from Mouse Prostate Stem Cells....................................................
Experience Summary .......................................................................................................
Organoid Citations ...........................................................................................................
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Organoid Culture Handbook
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Introduction to Organoid Culture
Organoids are organ-like structures that can be formed by 3D cell culture and differentiation of stem cells or
organ progenitors; and are capable of recapitulating aspects of organ function in vitro. Research & therapeutic
potential of organoids includes:
99
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Organogenesis Models
Drug Testing
Tumor, Disease and Infection Models
Toxicity Screening
Personalised Medicine
Regenerative Medicine / Organ Replacement
Preclinical models are falling short in predicting biological responses because plastic is not a natural
component and mouse models are not representative of the human body. These models fail to
recreate the complexity and the specificity of living tissues.
To recreate In vivo structure and function the following factors are necessary to consider:
In vivo structure and function: Factors to consider
Organoid Culture Recreates In Vivo Structure and Function
Tissue Resident Cells (Types, Quantities,
Organization)
Organoid Progenitor Cells (Differentiate into tissue-specific
cells)
Extracellular Matrix (Composition,
Organization, Compliance)
Extracellular Matrix (Basement Membrane Extract (BME))
Soluble Factors (Growth Factors, Cytokines)
Soluble Factors (Wnt, Noggin, EGF, R-Spondin-1, TissueSpecific Factors)
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Page | 3
Organoid Models
Organ
Brain
Retina
Salivary Glands
Esophagus
Lungs
Breast
Heart
Liver
Kidneys
Pancreas
Stomach
Small Intestine
Colon
Prostate
Page| 4
Images and Results
Citations and
Protocols (pg 23)
Pg 18, 19
3
Pg 19
Pg 13, 14
Pg 5, 15
Pg 16, 17
Pg 20, 21
Organoid Culture Handbook
1, 4, 10
1
7
2, 5, 7, 8
3, 6, 7
Featured Products for Organoid Culture
Organoid Progenitor Cells
Mouse Small Intestine Organoid Progenitor Cells
Organoid 3D cultures represent the next generation of tissue culture models. These cultures are extracted directly
from living tissues similar to primary cultures; however they are never subjected to an artificial, tissue culturetreated plastic environment. Instead, stem cell populations are maintained using a feeder-layer-free extracellular
matrix environment under non-differentiating conditions. When subjected to differentiating conditions, these
organoids exhibit expression of tissue-specific genes and differentiation of stem cells into tissue-specific
architecture.
The Cultrex® Organoid Progenitor Cells are derived from normal, healthy mouse small intestine tissue and are
continuously cultured using Basement Membrane Extracts, designed and qualified specifically to promote robust
organoid cultures (BME 2 and RGF BME R1). These organoid progenitor cells can be expanded and may be induced
to express tissue-specific markers under differentiating conditions.
A
B
Differentiation of Mouse Small Intestine Organoids Cultured on RGF BME R1. During expansion, Mouse Small
Intestine Organoids grow as spherical structures (A), but under differentiating conditions, crypt-like structures
bud from the organoid (B), mimicking intestinal epithelium. Nuclei were stained using DAPI (blue), and E-cadherin
was visualized via immunofluorescence (green).
Name
Cultrex® Organoid Progenitor Cells;
Mouse Small Intestine
Catalog Number
3750-001-01
www.amsbio.com | [email protected]
size
1 vial
Page | 5
Extracellular Matrices
BME (Basement Membrane Extract) for Organoid Culture
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Soluble form of basement membrane from Engelbreth-Holm-Swarm (EHS) tumor.
Major components include Laminin I, Collagen IV, Entactin & Heparan Sulfate Proteoglycan.
Gels at 37°C to form a reconstituted basement membrane
Higher concentration (14 -16 mg/ml)
Bigger Batches
Formulated for the Specific Task
Organoid qualified Matrix
BME can be used in a multiple applications, under a variety of cell culture conditions, for maintaining growth
or promoting differentiation of primary endothelial, epithelial, smooth muscle and stem cells. BME can also
be utilized in cell attachment, neurite outgrowth, angiogenesis, in vitro cell invasion and in vivo tumorigenicity
assays.
Name
Catalog Number
Pack size Buffer
Tensile
Strength
Recomended Applications
DMEM
High
Reduced growth factor
format optimized for robust
Organoid growth
DMEM
Higher
Organoid qualified (batch
tested for organoids)
suggested to use for difficult
to grow Organoid cultures
BME 2 Reduced Growth Factor (Organoid Matrix)
Cultrex® BME 2 (Reduced
Growth Factor Basement
Membrane Extract, Type 2,
Pathclear®)
3533-001-02
1ml
3533-005-02
5ml
3533-010-02
2 x 5ml
BME R1 Reduced Growth Factor
Cultrex® BME R1 (Reduced
Growth Factor Basement
Membrane Extract, Type
R1)
3433-005-R1
5ml
BME 2 provides a proprietary formulation that is higher in tensile strength when compared to our original BME,
making it more physiologically aligned with the in vivo tumor environment. It is difficult to know which matrix
formulation is best suited for any specific organoids, cell type, or application. However in competitive beta
tests, BME 2 consistently outperforms competitor products. Each BME lot is qualified on Human Small Intestine
Organoids.
The Growth Factor Reduced format (BME 2 RGF “organoid matrix”) has been shown to work well for growing
organoids, especially using techniques based on the LGR5 stem cell marker / Wnt signalling system, pioneered by
Hans Clevers and co-workers.
•
•
•
•
Each lot is validated for use in organoid culture
Used extensively for organoid culture
Reduced lot to lot variability
Easy to use
Recently we have developed an additional formulation of Cultrex® BME known as Cultrex® BME R1. This matrix
provides a proprietary formulation that has higher tensile strength when compared to our other products Cultrex®
BME, Cultrex® BME 2 and Cultrex® BME 3. It has a higher concentration of entactin, one of the BME components
that connects laminins and collagens reinforcing the hydrogel structure. Cultrex® BME R1 has been specifically
designed to culture tissue organoids.
Page| 6
Organoid Culture Handbook
Collagen I
Type I collagen is the major structural component of extracellular matrices found in connective tissue and
internal organs, but is most prevalent in dermis, tendons, and bone. It promotes cell attachment, proliferation,
differentiation, migration, and tissue morphogenesis. Cultrex® Rat Collagen I, Lower Viscosity is diluted to a lower
concentration (3 mg/ml); therefore it is less viscous and easier to handle.
Name
Cultrex® Rat Collagen I, Lower Viscosity
Cultrex® 3-D Collagen I (From Rat Tail Tendons)
Catalog Number
Pack size
3443-003-01
3443-100-01
1 ml @ 3 mg/ml
35 ml @ 3 mg/ml
3447-020-01
20 ml at 5mg/ml
Our 5 mg/ml rat collagen I (eg cat# 3447-020-01) is not pepsin treated, so it has intact telopeptides and is highly
viscous. It is provided in acetic acid and requires pH neutralization prior to use. Do not to attempt to pipet this
material using a graduated 1 ml pipette or narrow bore tip. Rather, we recommend using blue pipette tips (2001000uL capacity) that are clipped with sterile scissors to create a wide bore. Also, please consult the provided
product data sheet for pH neutralization instructions. We also offer 3 mg/ml Cultrex® Rat Collagen I, Lower
Viscosity (cat#s 3443-003-01 & 3443-100-01).
Our 3D Collagen has been tested extensively for the ability to promote growth and differentiation of cell types,
visualized by morphology in three dimensions in vitro (however, addition of laminin may be required as shown,
for MCF-10A cells, in the product data sheet).
Reagents
R-spondin (RSPO1) Expressing Cell Line
Roof plate-specific Spondin-1 (R-Spondin 1 or RSPO1), also known as CRISTIN3, is a 27 kDa secreted activator
protein that belongs to the R-Spondin family. R-Spondins positively regulate Wnt/beta-catenin signaling, most
likely by acting as a ligand for LGR4-6 receptors and an inhibitor for ZNRF3. R-Spondin-1 induces proliferation of
intestinal crypt epithelial cells, increases intestinal epithelial healing, and supports intestinal epithelial stem cell
renewal. The 293T cell line is stably transfected to express murine Rspo1 with an N-terminal HA epitope tag and
fused to a C-terminal murine IgG2a Fc fragment. This cell line is used to produce either purified Rspo1 or Rspo1
conditioned media. The murine Rspo1 protein has been used extensively in organoid culture to maintain Lgr5+
stem cells, and the FC and HA tags make it easy to purify or characterize.
Production of R-Spondin1 for organoid culture. The 293T cell line is stably transfected to express murine Rspo1
with an N-terminal HA epitope tag and fused to a C-terminal murine IgG2a Fc fragment. A) The HA-R-Spondin1Fc 293T cell line is cultured with zeocin to select for stably transfected cells. B) Production of HA-R-Spondin1-Fc
is characterized using Western Blot for R-Spondin1 protein (arrow). C) HA-R-Spondin1-Fc induces activation of
Wnt/ß-catenin response when evaluated using the Top-Flash Luciferase assay.
www.amsbio.com | [email protected]
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BENEFITS
•Cell line expresses recombinant mouse RSPO1 protein
•Positively regulates Wnt/beta-catenin signaling
•Essential medium component for most organoid culture models
•Purified protein and conditioned medium from our cell line has been used for culturing both human and mouse
organoids.
Name
Cultrex® R-spondin1 (RSPO1) Cells
Catalog Number
3710-001-01
size
1 vial (0.5 ml), 1x106 cells
Proteins and Antibodies
The Wnt family of secreted glycoproteins is involved in several important cell functions such as cell proliferation,
migration, polarity, survival, self renwal and cell fate. Among the most studied is a canonical Wnt signaling pathway
that regulates quantity of transcriptional co-activator β-catenin and in turn β-catenin then regulates expression
of the key developmental genes. In adition Wnt signals play an important role in the ability of organoids to
expand. In particular Wnt3a and RSPO1 (which acts as an Lgr receptor agonist) are two important factors used for
organoid expansion. AMSBIO offers a range of RSPO1 and Wnt3a recombinant human and mouse proteins. These
human recombinant proteins are purified from HEK293 cells while the mouse proteins are expressed in CHO cells.
Both are suitable for various cell assays and treatments.
R-spondin (RSPO1) Purified Proteins
Name
Catalog Number
size
Human R-Spondin 1 / RSPO1 Protein
AMS.RS1-H4221-50UG
50 μg
Human R-Spondin 1 / RSPO1 Protein
AMS.RS1-H4221-1MG
1 mg
Mouse R-Spondin 1 / RSPO1 Protein
AMS.RS1-M5220-50UG
50 μg
Mouse R-Spondin 1 / RSPO1 Protein
AMS.RS1-M5220-1MG
1 mg
Name
Catalog Number
size
Human Recombinant Wnt3a (75% purity)
AMS.rhW1HEKL-001
1 μg
Human Recombinant Wnt3a (75% purity)
AMS.rhW1HEKL-002
2 μg
Human Recombinant Wnt3a (75% purity)
AMS.rhW1HEKL-010
10 μg
Human Recombinant Wnt3a (85-90% purity)
AMS.rhW1HEKH-001
1 μg
Human Recombinant Wnt3a (85-90% purity)
AMS.rhW1HEKH-002
2 μg
Human Recombinant Wnt3a (85-90% purity)
AMS.rhW1HEKH-0010
10 μg
Mouse Recombinant Wnt3a (75% purity)
AMS.rmW3aL-001
1 μg
Mouse Recombinant Wnt3a (75% purity)
AMS.rmW3aL-002
2 μg
Mouse Recombinant Wnt3a (75% purity)
AMS.rmW3aL-010
10 μg
Mouse Recombinant Wnt3a (85-90% purity)
AMS.rmW3aH-001
1 μg
Mouse Recombinant Wnt3a (85-90% purity)
AMS.rmW3aH-002
2 μg
Mouse Recombinant Wnt3a (85-90% purity)
AMS.rmW3aH-010
10 μg
Wnt (Wnt3A) Proteins
Page| 8
Organoid Culture Handbook
Lgr5 (Gastric Stem Cell Marker) Antibodies
Name
Species Reactivity
Catalog Number
size
LGR5 mouse monoclonal antibody,clone UMAB212
Human
Human, Mouse
Human
Human, Mouse
UM800104
100 μl
UM800102
100 μl
UM800103
100 μl
TA503316
100 μl
LGR5 mouse monoclonal antibody, clone OTI7F2
(formerly 7F2)
Human, Mouse
TA808752
100 μl
LGR5 mouse monoclonal antibody, clone OTI3F1
(formerly 3F1)
Human, Mouse
TA808748
100 μl
LGR5 mouse monoclonal antibody,clone UMAB210
LGR5 mouse monoclonal antibody,clone UMAB211
LGR5 mouse monoclonal antibody, clone OTI2A2
Organoid Harvesting Solution
Organoid cultures exhibit cellular behaviors and
morphologies similar to those seen in vivo. However,
the adaptation of these models for studying
biochemical processes has been impeded by the
challenge of separating intact organoids from extracellular proteins comprising the hydrogel. Commonly,
proteases are employed to degrade these extracellular
proteins, however, proteases also degrade proteins
on the cell surface and protease activity may carry
over into subsequent cultures or lysate preparations.
Trevigen’s Cultrex® Organoid Harvesting Solution
provides a ready-to-use, non-enzymatic method
for depolymerizing extracellular matrix proteins to
allow for harvesting of intact organoids for passaging,
cryopreservation, or biochemical analysis.
PROTOCOL
BENEFITS
• Ready-to-use
• Non-enzymatic chelating solution
• Depolymerizes basement membrane matrix for
harvesting organoids from culture.
• Gentle for cells: preserves original morphology
APPLICATIONS
• Organoid passaging
• Sample preparation (PCR, Western Blot, and
Immunohistochemistry)
RESULTS
• See example results on page 22.
Name
Catalog Number
size
Cultrex® Organoid Harvesting Solution
3700-100-01
100 ml
www.amsbio.com | [email protected]
Page | 9
Organoid Culture Protocols
General Submerged Method for Organoid Culture
Developed by the Hans Clevers Lab, Hubrecht Institute, Netherlands
Citation:
Sato, T., R. G. Vries, et al. (2009). “Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal
niche.” Nature 459(7244): 262-265.
Centrifuge cells.
Such as: Cultrex® Organoid
Progenitor Cells; Mouse Small
Intestine (3750-001-01)
Add 50 µl to a 24
well plate
Add Culture Medium
Containing:
HA-R-spondin1-FC (from
3710-001-01)
Organoid
growth
Re-suspend cells in
organoid appropriate ECM:
1- BME 2 RGF (3533-010-02)
2- BME R1 (3433-005-R1)
Aspirate
Organoid
Culture Medium
and wash with
cold (4 °C) PBS
Crypt Organoid Culture Techniques
Crypt Isolation
Page| 10
Add cold (4 °C)
Organoid Harvesting
Solution (3700-100-01) –
30 min with gentle rocking
Organoid Culture Handbook
BME
depolymerizes
leaving intact
organoids
Organoids from Crypts
Air Liquid interface (ALI) Method for Organoid Culture
Developed by the Calvin Kuo Lab, Stanford University, USA
Citation:
– Ootani, A., X. Li, et al. (2009). “Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell
niche.” Nat Med 15(6): 701-706.
Organoids from Tissue
www.amsbio.com | [email protected]
Page | 11
ALI Method
Clonal Organoids from lgr5+ cells
Page| 12
Organoid Culture Handbook
Organoid Culture Examples
Liver Organoid Culture
Human Liver
Marker Expression in Human Liver Organoids on
BME 2 RGF.
Image courtesy of Helmuth Gehart/Professor
Hans Clevers, Hubrecht Institute, Utrecht,
Netherlands
In their paper in Cell, Huch, Clevers et al (2015)
demonstrated that:
• Primary human bile duct cells can readily be
expanded into 3D liver organoids in vitro using
BME 2 RGF
• Adult liver stem cells maintain self-renewal
capacity, differentiate into functional hepatocytes
in vitro and generate bona fide hepatocytes upon
in vivo transplantation.
• Expanded cells preserve their genetic integrity
over months in culture (agreeing with the authors’
previous observations in a mouse model).
• Organoids derived from patients with genetic
disorders can be used to model liver disease in
vitro.
Differential interference contrast image of human organoids grown in BME 2 RGF and cultured for >2
months in human liver complete medium. Magnification, 4X.
Image courtesy of Meritxell Huch, Gurdon Institute, University of Cambridge, UK
Long-term culture of Human Liver Organoids on BME 2 RGF. (Clonal cultures obtained by seeding sorted cells at
one cell per well)
Image courtesy of Meritxell Huch, Gurdon Institute, University of Cambridge, UK
www.amsbio.com | [email protected]
Page | 13
Marker Expression in Human Liver Organoids on BME 2 RGF.
Confocal image stained for ECAD (green) and the hepatocyte
marker HNF4 (red); nuclei counter-stained with Hoechst
(Blue).
Image courtesy of Dr. Meritxell Huch, Gurdon Institute,
University of Cambridge, UK
“We have obtained culture conditions that
allow us to long-term expand genetically
stable human donor liver cells in organoid
culture. One of the clues to this success
is the use of ECM that allows the cells to
grow in 3D. BME 2 has been our ECM of
choice for these experiments.”
Dr. Meritxell Huch (Gurdon Institute,
University of Cambridge, UK)
Differentiation of Organoids into
Hepatocytes on BME 2 RGF.
Expression of hepatocyte genes
in human liver organoid after 11
days on differentiation medium.
Immunofluorescence for albumin
(ALB, red) and ZO-1 (green); nuclei
counterstained with Hoechst (Blue)
Image courtesy of Dr. Meritxell
Huch, Gurdon Institute, University of
Cambridge, UK
Hepatocellular Carcinoma (HCC)
Hepatocellular carcinoma (HCC) organoid model,
grown in BME 2 RGF.
Image courtesy of Prof. Dr. med. Markus Heim,
Hepatology Group, Department of Biomedicine,
University Hospital Basel, Basel, Switzerland.
Page| 14
Organoid Culture Handbook
Mouse Liver
GFP-labeled organoids, grown from mouse liver stem
cells. grown on BME 2 RGF.
The cells were infected with a lentivirus expressing GFP
Image courtesy of Dr. Derk ten Berge, Erasmus MC
Stem Cell Institute, Rotterdam, Netherlands
Gastrointestinal Organoid Culture
Small Intestine Organoids
Small intestine organoids with DNA in blue
and structural proteins in red, grown on
Cultrex BME R1, reduced growth factor.
Images courtesy of J Wosen, E Mellins Lab,
Stanford University, USA.
www.amsbio.com | [email protected]
Page | 15
Human Colorectal
Human Colorectal Cancer (CRC)
organoids grown from single cells
on BME 2 RGF.
Immunofluorescence for Phalloidin
(red) to mark actin filaments; and
E- cadherin (green) as epithelial
marker; nuclei counterstained with
DAPI (blue)
Images courtesy of the Batlle Lab,
IRB Barcelona, Spain
BF
IF
Human Colorectal Cancer (CRC) organoids
grown for 7 days in BME 2 RGF.
Brightfield (BF) and Immunofluorescence
for Phalloidin (red) to mark actin filaments;
and laminin (green); nuclei counterstained
with DAPI (blue). Laminin stains basement
membrane and mediates the attachment,
migration and organization of cells.
Images courtesy of the Batlle Lab, IRB
Barcelona, Spain
Page| 16
Organoid Culture Handbook
Mouse Adenoma
Images courtesy of the Batlle Lab, IRB Barcelona, Spain
Mouse Intestine - Colorectal Cancer Model
WILD TYPE
APC-DELETED
Intestinal organoids derived from normal and Apc-deleted mice. (Loss of the negative Wnt pathway regulator
APC occurs in the majority of colorectal cancers.)
Images courtesy of the Li Lab, Francis Crick Institute, London, UK
Transgenic Mouse
Organoids (passage 1) derived directly into BME 2
RGF. Organoids were split once (using mechanical
disruption) following derivation; and the image was
taken 3 days later.
Existing organoid culture transferred to BME 2 RGF.
Organoids were digested to single cells, which were
plated onto BME 2 RGF. The image was taken 6 days
after plating.
Organoids prepared from the intestinal crypts of transgenic mice.
Images courtesy of the Sansom Lab, Beatson Institute of Cancer Research, Glasgow, UK
www.amsbio.com | [email protected]
Page | 17
Esophageal Organoid Culture
The images below show Organoids grown from tumors derived from fresh
tissue samples. These images are showing cells taken from the lowest part of
the esophagus almost into the stomach.
Esophageal Tumor
Images courtesy of Dr Mathew Garnett, Sanger Wellcome Trust Institute, Cambridge, UK
Esophageal Barrett’s Epithelial
Barrett’s esophagus is a precancerous condition where the esophageal lining becomes similar to the tissue
architecture of the intestine, it can develop following long term cases of gastro-esophageal reflux disease (GERD).
Images courtesy of Dr Mathew Garnett, Sanger Wellcome Trust Institute, Cambridge, UK
Page| 18
Organoid Culture Handbook
Esophageal Normal
Images courtesy of Dr Mathew Garnett, Sanger Wellcome Trust Institute, Cambridge, UK
Esophageal Normal Gastric
Images courtesy of Dr Mathew Garnett, Sanger Wellcome Trust Institute, Cambridge, UK
Breast Organoid Culture
Mammary cancer organoid from
mice, grown on / invading into BME
2 RGF.
Image courtesy of Tuula Kallunki,
Danish Cancer Society Research
Centre, Copenhagen, Denmark
www.amsbio.com | [email protected]
Page | 19
Prostate Organoid Culture
Metastatic Prostate Cancer-Derived Organoids
BME 2 (RGF)
Confocal images of
metastatic
prostate
cancer (mCRPC)-lymph
node biopsy grown
derived organoids grown
side-by-side on either
BME 2 (RGF) or BME R1
(RGF) for comparison.
Images courtesy of Dr
Veronica Gil, De Bono
Lab, ICR, Sutton, UK
BME R1 (RGF)
Harvested Organoids
These images on the left belong to same tumor biopsy-derived organoids
from the pictures above where growth and morphology was compared
between BME 2 and BME R1. These images are post-first passage using
Organoid Harvesting Solution and replating them.
The viability was very good and organoid proliferation rate has not been
affected by the solution and replating procedure, showing that the
reagent is effective.
Images courtesy of Dr Veronica Gil, De Bono Lab, ICR, Sutton, UK
Page| 20
Organoid Culture Handbook
Organoids from Mouse Prostate Stem Cells
Brightfield
YFP-labeled organoids, grown from
mouse prostate stem cells, grown on
BME 2 RGF.
The prostate cells (from Rosa26
-LstopL-YFP mouse) were infected
with Adenovirus expressing Cre to
activate YFP expression.
Images courtesy of the Baena
lab, Prostate Oncobiology, CRUK
Manchester Institute, Manchester,
UK
Alexa 488 (YFP)
www.amsbio.com | [email protected]
Page | 21
Experience Summary
Baena lab, Prostate Oncobiology
CRUK Manchester Institute, Manchester, UK ǀ p21
Batlle Lab
IRB Barcelona, Spain | pg 16 |17
Professor Hans Clevers
Hubrecht Institute, Utrecht, Netherlands | Helmuth Gehart pg 13 | Citations 1, 2, 4, 5, 6,
De Bono Lab
ICR, Sutton, UK | Dr Veronica Gil pg 20
Dr Mathew Garnett
Sanger Wellcome Trust Institute, Cambridge, UK | pg 18 | 19 | Citations 3, 6
Prof. Dr. med. Markus Heim
Hepatology Group, Department of Biomedicine, University Hospital Basel, Basel, Switzerland | pg 14
Dr. Meritxell Huch
Gurdon Institute, University of Cambridge, UK| pg 13| 14| Citations 1, 4
Tuula Kallunki
Danish Cancer Society Research Centre, Copenhagen, Denmark | pg 19
Li Lab,
Francis Crick Institute, London, UK | pg 17
E Mellins Lab
Stanford University, USA | J Wosen pg 15
Sansom Lab
Beatson Institute of Cancer Research, Glasgow, UK | pg 17
Dr. Derk ten Berge
Erasmus MC Stem Cell Institute, Rotterdam, Netherlands | pg 15
Page| 22
Organoid Culture Handbook
Organoid Citations
BME 2 RGF
1. Broutier, L., Andersson-Rolf, A., Hindley, C. J., Boj, S. F., Clevers, H., Koo, B. K., & Huch, M. (2016). Culture and
establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.
Nature Protocols, 11(9), 1724-1743.
2. Drost, J., Van Jaarsveld, R. H., Ponsioen, B., Zimberlin, C., Van Boxtel, R., Buijs, A., ... Korving, J. … & Clevers, H. (2015).
Sequential cancer mutations in cultured human intestinal stem cells. Nature, 521(7550), 43-47.
3. Francies, H. E., Barthorpe, A., McLaren-Douglas, A., Barendt, W. J., & Garnett, M. J. (2016). Drug Sensitivity Assays of
Human Cancer Organoid Cultures. Methods in molecular biology (Clifton, NJ)
4. Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M. M., ... & Clevers, H. (2014) Long-Term Culture
of Genome-Stable Bipotent Stem Cells from Adult Human Liver. Cell , 160, 299 – 312
5. Schwank, G., & Clevers, H. (2016). CRISPR/Cas9-Mediated Genome Editing of Mouse Small Intestinal Organoids.
Gastrointestinal Physiology and Diseases: Methods and Protocols, 3-11.
6. van de Wetering, M., Francies, H.E., Francis, J.M., Bounova, G., Iorio, F., Pronk, A., M., Garnett M.J., & Clevers, H. (2015)
Prospective derivation of a ‘Living Organoid Biobank’ of colorectal cancer patients. Cell 161(4), 933-945
Cultrex ® Rat Collagen I, Lower Viscosity
7. Li, X., Ootani, A., & Kuo, C. (2016). An Air–Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary
Gastrointestinal Tissues. Gastrointestinal Physiology and Diseases: Methods and Protocols, 33-40.
Published Protocols that have been used successfully with
BME 2 RGF matrix for organoid growth
8. Andersson-Rolf, A., Fink, J., Mustata, R. C., & Koo, B. K. (2014) A Video Protocol of Retroviral Infection in Primary
Intestinal Organoid Culture. JoVE (Journal of Visualized Experiments), (90), e51765-e51765.(‘Basement Matrix Extract
(Cultrex PathClear BME Reduced Growth Factor Type 2, 3533-005-02) supplied by AMSBIO can be used as an alterntive’see materials list).
9. Procedure for subculturing normal human gastric organoids, derived from the submerged method as described in
Barker, N., et al Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro. Cell Stem
Cell 6(1), 25–36
10.
Huch, M., Dorrell, C., Boj, S. F., van Es, J. H., Li, V. S., van de Wetering, M., ... & Haft, A. (2013) ). In vitro expansion of
single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature, 494(7436), 247-250
11.
Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., ... & Clevers, H. (2009) Single Lgr5
stem cells build crypt villus structures in vitro without a mesenchymal niche. Nature, 459(7244), 262-265
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Front Cover images courtesy of Dr. Meritxell Huch, Gurdon Institute, University of Cambridge, UK, the Batlle lab, IRB
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