Embed Size (px)
Citation preview
Organoid 3-D Culture
Gabriel Benton, Ph.D.
AACR Annual Meeting
New Orleans, LA
April 18, 2016
Objectives
• Provide background and rationale for organoid cultures
• Describe two of the common methodologies
• Offer tips for optimizing the procedures
• Describe ongoing projects at Trevigen to improve organoid culture models
Company Background
• Located in Gaithersburg, MD
• World Wide Distribution• 20 Years in Business• Cultrex Product Areas
– ECM Proteins– Standardized Assays– Contract Research Services
• Other Product Areas– DNA Damage– Oxidative Stress– Apoptosis
Society Goal: Improve Public Health by Treating Disease
• Drug development process is inefficient– Average cost ~ $5,000,000,000 (Forbes).– Average time ~ 10-15 years (PhRMA)
• Improve drug discover and preclinical assessment– Reduce cost– Increase throughput– Improve efficacy
Source: Pharmaceutical Research and Manufacturers of America, Drug Discovery and Development: Understanding the R&D Process, www.innovation.org
Why Are Preclinical Models Falling Short in Predicting Biological Response?
• Plastic is not a natural component of the human body
• Mice are not human
These models fail to recreate the complexity and specificity of living human tissues.
=
Identify Factors that Are Necessary to Recreate In Vivo Structure and Function
• Tissue Resident Cells– Types– Quantities– Organization
• Extracellular Matrix– Composition– Organization– Compliance
• Soluble Factors– Growth Factors– Cytokines
Organoid Cultures Recreate In Vivo Structure and Function
• Organoid Progenitor Cells– Differentiate into tissue-
specific cells
• Extracellular Matrix– Basement Membrane
Extract (BME)
• Soluble Factors– Wnt– Noggin– EGF– R-Spondin-1– Tissue-Specific Factors
Crypt base columnar cell
Paneth cells
Enterocytes
Goblet cells
Wnt
Organoids Possess Great Potential
iPSCs
TissueBiopsy
Organoid
• Disease Modeling• Drug Discovery• Drug Screening
0
0.2
0.4
0.6
0.8
1
1.2
A B C D E F
AB
AC
AD AE
AF
BC
BD BE
BF
CD CE CF
DE
DF EF
AB
C
AB
D
AB
E
AB
F
BC
D
BC
E
BC
F
BD
E
BD
F
CD
E
CD
F
AB
CD
AB
CE
AB
CF
BC
DE
BC
DF
AB
CD
E
AB
CD
F
BC
DEF
AB
CD
EF
Tum
or
Org
ano
id V
iab
ility
Compound(s)
Precision Medicine
GeneEditing
Regenerate Tissue
Brain
Lungs
Liver
Colon
Small Intestine
Pancreas
Kidneys
Stomach
Heart
Retina
Salivary Glands
Many Organoid Models Exist
Prostate
Mammary
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)
Stable, Long-Term Cultures may be Established from a Single Cell
Types of Organoid Cultures
• Air-Liquid Interface (ALI)
– Developed by the Kuo Lab, Stanford University, USA
• Submerged Culture
– Developed by the Hans Clevers Lab, Hubrecht Institute, Netherlands
Crypt Domains
Villus Domains
Lumen
Organoid Culture: ALI Method
Villus Domain
Crypt Domain
OrganoidsTissue
Minced tissue is embedded in collagen-1 at air-liquid interface
60 mm dish
30 mm insert with permeable membrane
Reconstituted collagen
• Collagen type 1• 10X medium• Neutralization
buffer• Keep cold (ice)
Warm (37 °C) Crypt Culture
Medium• Ham’s F12• Fetal calf serum• Gentamicin• R-Spondin 1-Fc
(for adult tissues)• Test compounds
ALI MethodTissue
Mince tissues at 4 °C (0.3 mm3)
Warm (37 °C) to polymerize acellular layer
Warm (37 °C) to polymerize organoid layer
Organoids remain suspended above medium level
Organoid Culture: ALI Method• Utilizes minced tissues (stromal
elements)• All tissue handling done on ice with
cold solutions (4 °C)• Reconstituted Cultrex® Collagen type
1– Maintain at 4 °C on ice as liquid– Warm to 37 °C to polymerize
• Tissue dissection– Tissue size (≤ 0.3 mm3)– Do not allow tissues to dry (5 minutes)
• Crypt culture medium– Below embedded organoid level– Frequency of change (up to 7 days)
based on organoid density and supplement stability
• Passage organoid culture– Every 2-4 weeks– Collagenase gel digestion– Mechanical dissociation of crypts
Organoids are suspended within the air-liquid interface.
Isolated Crypts
Purified LGR5+ crypt-based columnar stem cells
Crypts or stem cells are submerged in BME-2 containing WENR
Villus Domain
Crypt Domain
Organoids
Organoid Culture: Submerged Method
Wash and mince at 4 °C
to 5 mm
Wash with cold PBS
until clear (10-20 times)
Incubate in cold 2 mM EDTA, PBS for 15-30
min on ice with gentle
shaking
Centrifugeand remove supernatant containing
cells
Crypt Isolation
Pipet up and down in cold
PBS to dissociate
crypts
Resuspend crypts in cold
culture medium and
count
Pass crypts through a 70 µm strainer
Isolate Tissue
Organoids from Crypts
Centrifuge crypts
Resuspend crypts in BME-2
• Advanced DMEM/F12 • Wnt• EGF• HA-R-spondin1-FC• Noggin• Tissue-Specific Factors
Warm (37 °C ) Crypt Culture
Medium
Add 50 µl to a warm (37 °C) 24 well plate and incubate
at 37 °C for 25 min
Culture at 37 °C for 5 to 14 days
• All tissue handling done on ice with cold solutions (4 °C)
• Epithelial culture (no stromal elements)
• BME-2– Maintain at 4 °C on ice as
liquid– Warm to 37 °C to
polymerize
• Tissue dissection– Tissue size (5 mm)– Do not allow tissues to dry
(limit time)
Organoid Culture: Submerged Method
Images courtesy of the Batlle Lab, IRB Barcelona
Human Colorectal Cancer OrganoidsImmunofluorescence for Ecadherin(green) as epithelial marker;nuclei counterstained with DAPI (blue)
Centrifuge crypts
Organoids are fragmented
Count organoids to determine split
for passaging
BME depolymerizes
Add cold (4 °C) Organoid Harvesting Solution – 30 min with gentle rocking
Pass organoids through a 20 ga needle
Aspirate Organoid Culture Medium and
wash with cold (4 °C) PBS
Resuspend crypts in BME
Add 50 µl to a pre-warmed (37 °C) 24
well plate; incubate at 37 °C for 25 min
Add 500 µl warm (37 °C) Organoid Culture Medium
5 – 14 Days Culture
Organoid Culture/Passaging
• Maintain organoid cultures
– Change medium every Monday, Wednesday and Friday
• Passage organoid cultures
– Remove crypts from BME-2
– mechanically dissociate into single-crypt domains
– transfer to fresh BME-2
– perform every 1–2 weeks with a 1:3 to 1:5 split ratio
Organoid Culture: Submerged Method
Images courtesy of the Batlle Lab, IRB Barcelona
Human Colorectal Cancer OrganoidsImmunofluorescence for Phalloidin(red) to mark actin filaments and nuclei counterstained with DAPI (blue)
100 µM
1 2 3
4 5Days
Mouse Colon Organoids Exhibit Budding Morphology
D0 D1 D2
D3 D4 D6
D8 D10 D11
Human Gastric Organoids Exhibit Spheroid Morphology
Organoid Differentiation
• Removal of factors that inhibit differentiation
– Wnt
• Addition of factors that promote differentiation
– DAPT (Notch pathway inhibitor)
• High culture densities
• Extended culture periods without passaging
-Wnt+5 µMDAPT
+WNER
Marker Expression in Human Liver Organoids onBME 2 (RGF). Confocal image stained for ECADand the hepatocyte marker HNF4. Nucleicounterstained with Hoechst. (Image courtesy ofMeritxell Huch, Gurdon Institute, University ofCambridge, UK)
Differentiation of Organoids into Hepatocytes (liver cells) onBME 2 (RGF). Expression of hepatocyte genes in human liverorganoid after 11 days on differentiation medium (DM).Immunofluorescence for albumin (ALB, red) and ZO-1 (green);nuclei counterstained with Hoechst (Blue) (Image courtesy ofMeritxell Huch, Gurdon Institute, University of Cambridge, UK)
Expression of Tissue-Specific Markers
ECM Development: Entactin-Rich BMEBME
+Entactin BME-2B
atch
1
Bat
ch 2
Bat
ch 3
Bat
ch 1
Bat
ch 2
Bat
ch 3
0
1
2
3
4
5
6
7
8
9
10
Entactin Rich BME BME-2
Perc
ent
Enta
ctin
(D
en
sito
met
ry)210
111
71
55
41
kDa
Entactin is an integral component of the basement membrane
• Structural linkage for laminin, collagen-4, and perlecan
• Cell adhesion molecule for αvβ3 and α3β1 integrins
Laminin
Entactin
Collagen-4
Perlecan
Cell Surface
Entactin Enrichment Improves Take for Gastric Organoids
Summary
• Organoid cultures recreate tissue architecture and gene expression ex vivo and in vitro using:– Tissue or stem cells
– Extracellular matrix
– Tissue-specific soluble factors
• Stem cells may be propagated using specific medium formulations (such as Wnt, EGF, Noggin, R-Spondin)
• Cells may be directed to differentiate into tissue-specific lineages using modified medium formulations
• ECM modification may enhance organoid culture
Organoid Culture ProductsCatalog # Product Name Size
3533-001-02 Cultrex® Reduced Growth Factor Basement Membrane Extract, Type 2, PathClear®
1ml
3533-005-02 Cultrex® Reduced Growth Factor Basement Membrane Extract, Type 2, PathClear®
5ml
3533-010-02 Cultrex® Reduced Growth Factor Basement Membrane Extract, Type 2, PathClear®
2x5ml
3700-100-01 Cultrex Organoid Harvesting Solution 100 ml
3710-001-01 Cultrex R-spondin1 (RSPO1) Cells 1 vial (0.5 ml),
1x10^6 cells
3-D Culture: Where In Vivo Meets In Vitro™We design, develop, and deliver matrices and kits for 3-D Culture.
Visit us at www.trevigen.com
Questions?Technical – [email protected] – [email protected]
