APPLICATION NOTEOPTIMIZING ORGANOID CULTURE CONDITIONS:THE IMPORTANCE OF GROWTH FACTOR
BIOACTIVITY AND REAGENT CONSISTENCYINTRODUCTION
An organoid is a miniature, simplified version of an organ produced in vitro that resembles the cellular composition and architecture of the tissue of origin and exhibits functional similarities.
Organoids are derived from primary tissue, embryonic stem cells(ESCs), or induced pluripotent stem cells (iPSCs), which are capable of self-renewal and differentiation. Under specific culture
conditions, these cells are driven to form 3-dimensional structures that self-organize into organ-like tissues. Organoids typically consist of multiple cell types that are correctly positioned withrespect to both each other and the extracellular matrix and arepresent in a physiologically relevant microenvironment. They arealso amenable to long-term expansion and manipulation. As aresult, they are being increasingly used as in vitro model systems
for studying human organ development, modeling disease conditions, screening for drug efficacy or toxicity, and investigating
personalized medicine.

BRAIN ORGANOIDS

INNER EARORGANOIDSPANCREATIC
ORGANOIDSLUNG
ORGANOIDSLIVER
ORGANOIDSKIDNEYORGANOIDSGASTROIDSENTEROIDS

Organoid systems have been developed for a variety of different
human tissues, including brain, colon, inner ear, intestine, kidney,
PROSTATE
liver, lung, pancreas, prostate, and retina, among others. Since
ORGANOIDS
COLONOIDS
the conditions for culturing different organoids are variable, thereagents and protocols needed for their generation have to betested and optimized. Once optimal culture conditions are established, researchers must ensure that these conditions are reproducible so that the organoids can be successfully passaged, and the researcher can be confident that they are working on identicalsystems from one experiment to the next.

R&D SYSTEMS® IS THE MOST TRUSTED SOURCE FOR
RECOMBINANT PROTEINS FOR ORGANOID RESEARCH
Some of the most important components of organoid media are
growth factors such as R-Spondins, Noggin, and Wnt-3a, which
need to display high levels of activity, batch-to-batch consistency,
and be free of contaminants to ensure that they provide optimal,
consistent organoid growth. Since these proteins can be difficult
to make, it is critical that these reagents are produced or obtained
from a reliable source to ensure that they maintain a high level ofactivity and consistency. This allows researchers to be confident
that their experiments will be reproducible over time.

ber of publications that cite the use of R&D Systems R-Spondins,
Noggin, or Wnt-3a proteins for culturing organoids. Our recombinant human R-Spondin 1, Wnt-3a, and Noggin consistently
show higher levels of bioactivity than competitors’ proteins andeach new lot is tested side-by-side with previous lots to ensurethat the new lot displays the same level of bioactivity and purity
as previous lots. New lots are also tested to make sure that theymeet our expectations for endotoxin specifications, which is anindustry-leading <0.1 EU/µg. With over 30 years of experiencepurifying and manufacturing recombinant proteins, we offer customers stability of source and the expertise necessary to ensurethat our growth factors provide superior performance and minimal lot-to-lot variability.

Many organoid researchers rely on R&D Systems as a trusted
source for these reagents due to our rigorous in-house testingand quality control specifications. This is evidenced by the num-

R&D SYSTEMS RECOMBINANT HUMAN R-SPONDIN 1, WNT-3A, AND NOGGIN DISPLAYHIGHER LEVELS OF ACTIVITY THAN LEADING COMPETITORS’ PROTEINSR&D Systems
Competitor

420000320000220000120000
10

-6

10

-5

10

-4

10

-3

10

-2

10

-1

10

0

10

1

C

2.0

R&D Systems
Top Competitor

1.5

1.0

0.5

10-2

10-1

100

101

R-Spondin 1 (μg/mL)

Wnt-3a (ng/mL)

102

103

Alkaline Phosphatase Production (Mean O.D.)

B

Alkaline Phosphatase Production (Mean O.D.)

Relative Luciferase Activity (Mean RFU)

A

1.2

R&D Systems
Top Competitor

1.0

0.8

0.6

0.4

0.2
10-2

10-1

100

101

102

Noggin (μg/mL)

R&D Systems Recombinant Human R-Spondin 1, Wnt-3a, and Noggin Display Higher Activity than the Same Proteins Provided by Leading Competitors. (A) The ability of
R&D Systems Recombinant Human R-Spondin 1 (Catalog # 4645-RS; blue line) or recombinant human R-Spondin 1 from a leading competitor (purple line) to stimulate
activation of beta-Catenin using a TOPflash beta-Catenin/TCF reporter assay was tested in the HEK293T human kidney cell line, in the presence of Recombinant Mouse
Wnt-3a (R&D Systems, Catalog # 1324-WN; 5 ng/mL). The ED50 for this effect for R&D Systems Recombinant Human R-Spondin 1 was approximately 7-fold greater than thecompetitor’s R-Spondin 1. We also offer highly active Recombinant Human R-Spondin 3 (R&D Systems, Catalog # 3500-RS), which is being increasingly used for culturing
different types of organoids. (B) The bioactivity of R&D Systems Recombinant Human Wnt-3a (Catalog # 5036-WN; orange line) or human Wnt-3a from another company(green line) was assessed by measuring the ability of the proteins to induce alkaline phosphatase production in the MC3T3-E1 mouse preosteoblast cell line. The ED50 for
this effect of R&D Systems Recombinant Human Wnt-3a was 1.7-fold better, with more than twice the maximum response compared to the competitor’s Wnt-3a. (C) The
bioactivity of R&D Systems Recombinant Human Noggin (Catalog # 6057-NG; orange line) or recombinant human noggin from a top competitor (green line) was assessedby measuring the ability of the proteins to inhibit alkaline phosphatase production induced by 50 ng/mL Recombinant Human BMP-4 (Catalog # 314-BP) in the ATDC5mouse chondrogenic cell line. The ED50 for this effect for R&D Systems Recombinant Human Noggin in the presence of 50 ng/mL of Recombinant Human BMP‑4 wasapproximately 30-fold greater than the top competitor’s Noggin.

2

R&D SYSTEMS RECOMBINANT HUMAN R-SPONDINS, WNT-3A, AND NOGGIN DISPLAY HIGH
LOT-TO-LOT CONSISTENCY TO SUPPORT OPTIMIZED, CONSISTENT ORGANOID GROWTH
17000
15000
13000
11000
9000
7000
5000
1e-4

0.001

0.01

0.1

1

10

100

1000

C
14000
12000
10000
8000
6000
4000

10000

1e-5

1e-4

0.001

0.01

0.1

1

10

100

R-Spondin 3 (ng/mL)

R-Spondin 1 (ng/mL)

1000

Alkaline Phosphatase Production (Mean O.D.)

B
Relative Luciferase Activity (Mean RFU)

Relative Luciferase Activity (Mean RFU)

A

1.5

1

0.5

0
0.001

0.01

0.1

1

10

100

1000

10000

Wnt-3a (ng/mL)

Lot-to-Lot Consistency Testing of Recombinant Human R-Spondin 1, R-Spondin 3, and Wnt-3a. Three independent lots of (A) Recombinant Human R-Spondin 1(R&D Systems, Catalog # 4645-RS), or (B) Recombinant Human R-Spondin 3 (R&D Systems, Catalog # 3500-RS) were tested for their ability to stimulate activation of betaCatenin using a TOPflash beta-Catenin/TCF reporter assay in the HEK293T human kidney cell line, in the presence of 5 ng/mL Recombinant Mouse Wnt-3a (R&D Systems,
Catalog # 1324-WN). Each trace shown on the graph represents data obtained from Recombinant Human R-Spondin 1 or Recombinant Human R-Spondin 3 from a different
manufacturing run. (C) Three independent lots of Recombinant Human Wnt-3a (R&D Systems, Catalog # 5036-WN) were tested for their ability to induce alkaline phosphataseproduction in the MC3T3-E1 mouse preosteoblast cell line. Each trace shown on the graph represents data obtained from Recombinant Human Wnt-3a from a different
manufacturing run.

Alkaline Phosphatase Production (O.D.)A

B

R&D Systems

2Competitor 2

1.5

1

0.5

0
1e-4

0.001

0.01

0.1

1

10

100

1000

350

10000

SOX1+ Clusters

Noggin (μg/mL)

300
250
200
150
100
50
0

No
Competitor
Noggin1Competitor
2

Lot 1

Lot 2

R&D Systems

Lot-to-Lot Consistency Testing of Recombinant Human Noggin Demonstrates that R&D Systems Noggin Protein Displays Minimal Lot-to-Lot Variability and Higher Activity
than Two Leading Competitors’ Noggin Proteins. (A) Three independent lots of Recombinant Human Noggin (R&D Systems, Catalog # 6057-NG) were tested for their ability
to inhibit alkaline phosphatase production induced by 50 ng/mL Recombinant Human BMP-4 (R&D Systems, Catalog # 314-BP) in the ATDC5 mouse chondrogenic cell line.
Each trace shown on the graph (orange, light blue, purple) represents data obtained from Recombinant Human Noggin from a different manufacturing run. The blue traceshows the ability of Recombinant Human BMP-4 (50 ng/mL) to induce alkaline phosphatase production. (B) BG01V human embryonic stem cells were cultured in Mouse
Embryonic Fibroblast Conditioned Media (R&D Systems, Catalog # AR005) supplemented with FGF basic (5 ng/mL). Stem cells were driven into early cells of theneuroectoderm using a 3-day incubation in Recombinant Human Noggin (25 µg/mL) from either R&D Systems (Lot 1, Lot 2; Catalog # 6057-NG) or from two separate
competitors (Competitor 1, Competitor 2). Control cells were not incubated in Noggin (No Noggin). (Top) Representative images of embryonic stem cells that were stained
for the early ectoderm marker, Otx2 (red), the neuroectoderm marker, SOX1 (green), and DAPI (blue), following differentiation with Noggin from R&D Systems or Nogginfrom Competitor 2. (Bottom) Quantification of SOX1+ clusters under each of the indicated culture conditions. Cells treated with R&D Systems Noggin showed an increase
in SOX1+ cells compared to both untreated and competitor-treated cells. R&D Systems Noggin also showed consistent differentiation across the lots tested. BG01V humanembryonic stem cells are licensed from ViaCyte, Inc.

3

BIO-TECHNE® OFFERS A COMPLETE PORTFOLIO OF REAGENTS
DESIGNED TO PROMOTE THE ROBUST, REPRODUCIBLE
EXPANSION OF ORGANOIDS
Basement Membrane Extract (BME), Cultrex Organoid Harvesting Solution, media, extracellular matrix proteins, N-2 MAX and
N-21 MAX media supplements, and small molecules commonlyused in organoid cultures.

In addition to R&D Systems recombinant proteins, Bio-Techneoffers a range of other products that are designed and tested to provide superior performance and maintain consistency during the culture, expansion, and passaging of organoids.
These include different formats of organoid-qualified CultrexTM

CULTREX TM ULTIMATRIX REDUCED GROWTH FACTOR (RGF) BASEMENTMEMBRANE EXTRACT
al enhancements translate into substantial performance benefits
that make Cultrex UltiMatrix RGF BME an ideal extracellular matrix for consistent stem cell and organoid cell culture. View Our
Complete Selection of Organoid-Qualified Basement Membrane
Extracts | Cultrex BME & ECM Products

A number of different formats of Cultrex Basement Membrane Extract (BME) are available for 2-D and 3-D cell culture.
Cultrex UltiMatrix Reduced Growth Factor (RGF) BME is a soluble form of basement membrane that provides high tensile
strength, enhanced levels of entactin/nidogen, elevated protein
concentration, and robust clarity and purity. These composition-Lot:

1 2 1 21 2 1 2

440 -

440 -

280 -

200 -

66 -

Entactin

Lot:

280 -

280 -

200 -

200 -

1 2 1 2

116 -

66 -

66 -

kDa

kDa

Laminin 1!" #$ " #" " #$ %#" %#$ &#" &#$ 40

120

440 -

116 -

116 -

kDa

Lot:

B

ME
trix B BME
ltiMa
rex U Alternate
Cult

110

35

@A*6 ( .+B- C/DE+F( */D*- (

100
90

30

80

25

70
60
50

Alternate BME Lot #1

20

Alternate BME Lot #2

15

40
Ultimatrix Lot #1

30

10

Ultimatrix Lot #2

20

5

10

Temperature0Collagen IV0α, β chains

Temperature (°C)

ME
trix B BME
ltiMa
rex U Alternate
CultME
trix B BME
ltiMa
rex U Alternate
Cult

Storage modulus (G’) (Pa)

A

100

200

300

400

500

600

700

800

900

0

Time (sec)Cultrex UltiMatrix RGF Basement Membrane Extract (BME) Displays Consistent Lot-to-Lot ECM Composition, High Tensile Strength, and Gel Rates. A) Simple Western
analysis for Entactin, Laminin, and Collagen IV across two lots of both Cultrex UltiMatrix RGF BME (R&D Systems, Catalog # BME001) and an alternate commercial BME
matrix. Cultrex UltiMatrix RGF BME shows consistent expression of Entactin, compared to the alternate ECM matrix. B) The tensile strength (elastic modulus) dynamics across
two lots of Cultrex UltiMatrix RGF BME and an alternate commercial BME matrix. The elastic modulus was measured at increasing temperatures to determine tensile strengthand gelling rate across two independent lots of Cultrex UltiMatrix RGF BME (R&D Systems, Catalog # BME001) and the alternate BME matrix. Cultrex UltiMatrix RGF BME
was found to display consistent gelling rates and high tensile strength (storage modulus) compared to the alternate commercial BME matrix.

A

B

C

Human Descending Colon Organoids Grown in Cultrex UltiMatrix RGF Basement Membrane Extract. Adult stem cells isolated from human descending colon wereembedded in Cultrex UltiMatrix RGF BME (R&D Systems, Catalog # BME001) and cultured in growth medium for 30 days prior to imaging. A) Brightfield image of descending
colon organoid showing tissue invagination and epithelial layer formation. B, C) Descending colon organoids were stained with an Anti-Human Chromogranin A Monoclonal
Antibody (green; R&D Systems, Catalog # MAB90981), to visualize intestinal enteroendocrine cells, and counterstained with an Anti-Human/Mouse E-Cadherin Antigen
Affinity-purified Polyclonal Antibody (red; R&D Systems, Catalog # AF748) and DAPI (blue; R&D Systems, Catalog # 5748).

4

SERUM-FREE MEDIA SUPPLEMENTS
N-2 MAX and N21-MAX media supplements are also available
to support organoid cultures. The N21-MAX Media Supplement
improves upon the traditional B27- and NS21-like supplements,
which are being increasingly used to optimize stem cell and 3-D
cell culture media. Both the N21-MAX and N-2 MAX media supplements are serum-free, fully-defined supplements that are deB% Nestin-positive Cells% SOX2-positive Cells

100
80
60
40
20
0
Lot 1

Lot 2

Lot 3

Lot 4

C
100

100

80

80

% Positive Cells

A

signed to eliminate uncontrolled variables, such as those foundin serum-containing media and in some commercial serum-free
supplements. These media supplements reduce unwanted experimental variations and have been shown to improve stem celldifferentiation and increase the viability and health of differentiated cell types during long-term cell culture conditions.

60
40
20
0

Lot 5

Lot 1

Lot 2 Lot 3

Lot 4 Lot 5

60
40
20
0
SOX2

NestinLot-to-Lot Consistency Testing of N-2 MAX Media Supplement. Rat cortical stem cells (RCSC) were cultured over multiple passages using N-2 MAX (R&D Systems, Catalog# AR009) and evaluated via flow cytometry for the maintenance of the stem cell markers, SOX2 and Nestin. Quantification of flow cytometry data demonstrates a high purityof (A) SOX2 or (B) Nestin positive RCSCs across multiple lots of N-2 MAX Supplement. (C) Average SOX2 and Nestin-positive RCSCs, demonstrate the consistent lot-to-lot
performance of N-2 MAX.

SMALL MOLECULES FOR CULTURING
ORGANOIDSCULTREX ORGANOID HARVESTING
SOLUTION

Bio-Techne also offers a wide range of Tocris® small moleculesthat are commonly used as 3-D growth matrix components forgenerating different types of organoids and sustaining long-term
growth. These include products such as A 83-01, N-acetylcysteine, CHIR 99021, DAPT, dexamethasone, Gastrin I, nicotinamide,
SB 202190, and more.

Our catalog also includes Cultrex Organoid Harvesting Solution,
which provides a non-enzymatic method for depolymerizing extracellular matrix proteins, allowing intact organoids to be harvested for passaging, cryopreservation, or biochemical analysis.

CRYOPRESERVATION MEDIA
Protein-free cryopreservation media for optimized cryopreservation of cell lines or cryopreservation of stem cells are also available.
These specially formulated media contain a defined serum substitute as well as an optimized concentration of a cryopreservative that
increases the recovery and viability of healthy cells compared to conventional freezing media.

ANTIBODIES AND RNASCOPE ® IN SITU HYBRIDIZATION ASSAYS
Beyond our portfolio of reagents for organoid culture, we also offer a large selection of R&D Systems® antibodies andNovus Biologicals® antibodies for identifying lineage-specific markers, as well as RNAScope® in situ hybridization assays for detecting
specific target RNAs in intact cells, when appropriate antibodies are not available.
Our research is greatly facilitated by Bio-Techne products. The various organoid systems and co-cultures are all performed inor on R&D Systems Cultrex reduced growth factor BME with great results. Even for more exotic organoid, such as snake venom
gland organoids, we could achieve breakthroughs with R&D Systems growth factors and BME as well as spatial visualization of
toxin transcripts using ACD’s RNAScope .
Jens Puschof, Hans Clevers lab, Hubrecht Institute, The Netherlands.

5

R&D SYSTEMS SCIENTISTS ARE DEVELOPING ORGANOID CULTURE PROTOCOLS AND
OTHER ORGANOID-RELATED RESOURCES TO ASSIST RESEARCHERS
To further assist researchers that are working on generating organoids as model systems, scientists at R&D Systems are also
working hard to optimize the culture conditions for growing different types of organoids. As we see success, we are publishing
the lists of the reagents that we used, along with the recipes andprotocols in our Organoid Resource Database, where they canbe easily accessed by all researchers interested in utilizing organoids as model systems. As an example, the materials needed to
culture human liver or lung organoids are shown below with links
to our in-house protocols. In addition to these protocols, our organoid-related webinars, blogs, scientific posters, and literature
are also available on our Organoid Resources page.

CULTURING HUMAN LIVER ORGANOIDSTABLE 1. Materials needed for human liver organoid culture
REAGENT NAME

SUPPLIER

CATALOG #REAGENT NAME

SUPPLIER

CATALOG #Cultrex Organoid Harvesting
Solution

R&D Systems

3700-100-01

Recombinant Human EGF

R&D Systems

236-EG

Recombinant Human R-Spondin 1

R&D Systems

Cultrex UltiMatrix Reduced Growth
Factor Basement Membrane Extract

R&D Systems

BME001-05

4645-RS

Recombinant Human Noggin

R&D Systems

6057-NG

Advanced DMEM/F-12 Cell Culture
MediumThermo Fisher

12634-010

Recombinant Human FGF-10

R&D Systems

345-FG

Recombinant Human FGF-19

R&D Systems

Glutamine

Tocris Bioscience

969-FG

5823

Recombinant Human BMP7

R&D Systems

HEPES

354-BP

Tocris Bioscience

3173

Recombinant Human HGF

R&D Systems

294-HG

N21-MAX Supplement

R&D Systems

AR008

Forskolin

Tocris Bioscience

1099

N-2 MAX Supplement

R&D Systems

AR009

A 83-01 (ALK5 inhibitor)

Tocris Bioscience

2939

N-Acetylcysteine

Tocris Bioscience

5619

Recombinant Human Wnt-3a

R&D Systems

5036-WN

Gastrin I (Human)

Tocris Bioscience

3006

DAPT

Tocris Bioscience

2634

Nicotinamide

Tocris Bioscience

4106

Dexamethasone

Tocris Bioscience1126

Y-27632 dihydrochloride (Rho
Kinase Inhibitor)

Tocris Bioscience

1254

View the complete Human Liver Organoid Culture protocol available in our Organoid Resources Database.
A

B

C

A

Undifferentiated and Differentiated Human Liver Organoids. Representative brightfield images of human A) undifferentiated and B) differentiated liver organoids that were
cultured using Cultrex UltiMatrix RGF Basement Membrane Extract (R&D Systems, Catalog # BME001) and the Bio-Techne reagents listed in the Materials table shown above
for culturing human liver organoids. The organoids were differentiated in media containing Recombinant Human FGF-19 (R&D Systems, Catalog # 969-FG), DAPT
(Tocris, Catalog # 2634), and Dexamethasone (Tocris, Catalog # 1126). C) Undifferentiated liver organoids shrink as they differentiate and have positive expression of thehepatocyte markers, Albumin (C, top) and HNF-3beta (C, bottom), which were detected using a Mouse Anti-Human Serum Albumin Monoclonal Antibody (top, red;
R&D Systems, Catalog # MAB1455) and a Goat Anti-Human HNF-3beta Antigen Affinity-purified Polyclonal Antibody (bottom, red; R&D Systems, Catalog # AF2400),
respectively. DAPI (R&D Systems, Catalog # 5748) was used as a counterstain in part C of the figure (blue).

6

CULTURING HUMAN LUNG ORGANOIDSTABLE 1. Materials needed for human lung organoid culture
REAGENT NAME

SUPPLIER

CATALOG #A 83-01

Tocris Bioscience

2939

Cultrex Organoid Harvesting
Solution

R&D Systems

3700-100-01

Cultrex UltiMatrix Reduced
Growth Factor Basement
Membrane Extract

R&D Systems

BME001-05

Advanced DMEM/F-12 Cell
Culture Medium

Thermo Fisher

12634-010

Glutamine

Tocris Bioscience

5823

HEPES

Tocris Bioscience

3173

N21-MAX Supplement

R&D Systems

AR008

N-Acetylcysteine

Tocris Bioscience

5619

Penicillin/Streptomycin

R&D Systems

B21210SB 202190 (p38 MAPK Inhibitor)

Tocris Bioscience

1264

Nicotinamide

Tocris Bioscience4106

Y-27632 dihydrochloride (Rho
Kinase Inhibitor)

Tocris Bioscience

1254

Recombinant Human R-Spondin 1

R&D Systems

4645-RS

Recombinant Human Noggin

R&D Systems

6057-NG

Recombinant Human FGF-10

R&D Systems

345-FG

Recombinant Human FGF-7

R&D Systems

251-KGThe complete Human Lung Organoid Culture protocol available in our Organoid
Resources Database.
A

B

C

Characterization of Human Lung Organoids. Representative images of human lung organoids for tissue-specificcell types. A) Expression of SOX2 (green) and Acetylated Tubulin (red) was assessed using an Anti-Human/Mouse/
Rat SOX2 Antigen Affinity-purified Polyclonal Antibody (R&D Systems, Catalog # AF2018) and an Anti-Acetylated
Tubulin Monoclonal Antibody (Novus Biologicals, Catalog # NB600-567), respectively. B) Expression of p63/
TP73L (green) and Cytokeratin 10 (green) was assessed using an Anti-Human p63/TP73L Antigen Affinity-purified
Polyclonal Antibody (R&D Systems, Catalog # AF1916) and an Anti-Cytokeratin 10 Monoclonal Antibody(Novus Biologicals, Catalog # NBP2-61736), respectively. C) Expression of Podoplanin (green), a marker of type 1
alveolar cells, was assessed using an Anti-Human Podoplanin Antigen Affinity-purified Polyclonal Antibody(R&D Systems, Catalog # AF3670).

To view our complete collection of Organoid-related webinars, blogs, scientific posters,
and literature, please visit our Organoid Resources Database webpage.

7

Human Lung Organoids Grown in Cultrex UltiMatrix
RGF
Basement
Membrane
Extract(BME).
Representative images of lung organoids, derived
from lung biopsy adult stem cells, embedded in
Cultrex UltiMatrix RGF BME (R&D Systems, Catalog #BME001) and cultured in Lung Organoid Expansion
Media. Images show organoids at day 52 of culture.

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