A Comprehensive Guide to Organoid Culture

Introduction

Organoids are organ-like structures that are produced by three-dimensional (3D) cell culture and differentiation of organ progenitors or stem cells. These structures can recapitulate the aspects of organ function in vitro. The following are the research and therapeutic potential of organoids:

  • Drug testing
  • Organogenesis models
  • Toxicity screening
  • Tumor, disease and infection models
  • Organ replacement / regenerative medicine
  • Personalized medicine

Figure 1. 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.

The following factors need to be considered to recreate in vivo structure and function:

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 (growth factors, cytokines)

Organoid progenitor cells

Mouse small intestine organoid progenitor cells

Representing the next generation of tissue culture models, organoid 3D cultures are directly extracted from living tissues just like primary cultures. However, these cultures are not exposed to an artificial, tissue culture-treated plastic environment, but a feeder-layer-free extracellular matrix environment is used to maintain the stem cell populations under non-differentiating conditions.

When these organoids are subjected to differentiating conditions, they show expression of tissue-specific genes and differentiation of stem cells into a tissue-specific architecture.

Derived from normal and healthy mouse small intestine tissue, the Cultrex® organoid progenitor cells are continuously cultured using basement membrane extracts (BMEs), which are specifically designed and qualified to promote robust organoid cultures (RGF BME R1 and BME 2). It is possible to expand and induce these organoid progenitor cells to express tissue-specific markers under differentiating conditions.

Figure 2. 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

Catalog number

size

Cultrex® organoid progenitor cells; mouse small intestine

3750-001-01

1 vial


Extracellular matrices

BME for organoid culture

  • Key components include collagen IV, laminin I, heparan sulfate proteoglycan, and entactin
  • Soluble form of basement membrane from Engelbreth-Holm-Swarm (EHS) tumor
  • Organoid qualified matrix
  • Gels at 37°C to produce a reconstituted basement membrane
  • Higher concentration (14 -16mg/ml)
  • Bigger batches
  • Formulated for the particular task

BME, under various cell culture conditions, can be used in many applications to maintain growth or promote differentiation of primary epithelial, endothelial, stem, and smooth muscle cells. It can even be utilized in angiogenesis, neurite outgrowth, cell attachment, in vitro cell invasion, and in vivo tumorigenicity assays.

Name

Catalog number

Pack size

Buffer

Tensile strength

Recommended applications

BME 2 reduced growth factor (organoid matrix)

Cultrex® BME 2 (reduced growth factor basement membrane extract, type 2, Pathclear®)

3533-001-02

1ml

DMEM

High

Reduced growth factor format optimized for robust organoid growth

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

DMEM

Higher

Organoid qualified (batch tested for organoids) suggested to use for difficult to grow organoid cultures

A proprietary formulation provided by BME 2 has a higher tensile strength compared to the original BME, This makes the former more physiologically aligned with the in vivo tumor environment. However, it is not easy to establish which matrix formulation is suitable for specific cell type, organoids, or application.

Conversely, BME 2 has been shown to be better than competitor products in competitive beta tests. All BME lots are qualified on human small intestine organoids.

It has been shown that the growth factor reduced format (BME 2 RGF “organoid matrix”) is suitable for growing organoids, particularly when using techniques based on the LGR5 stem cell marker/Wnt signaling system, developed by Hans Clevers and team.

  • Used widely for organoid culture
  • All lots are validated for use in organoid culture
  • Easy to use
  • Reduced lot to lot variability

An additional formulation of Cultrex® BME called Cultrex® BME R1 was recently developed that offers a proprietary formulation. When compared to other products such as Cultrex® BME 3, Cultrex® BME 2, and Cultrex® BME, this formulation has a higher tensile strength and a higher concentration of entactin, which is one of the BME components that couples collagens and laminins reinforcing the hydrogel structure. Cultrex® BME R1 was designed for growing tissue organoids.



Collagen I

Type I collagen is an important structural component of extracellular matrices present in internal organs and connective tissue; however, it is commonly found in bones, tendons, and dermis. Type I collagen promotes cell proliferation, attachment, migration, differentiation, and tissue morphogenesis.

The Cultrex® rat collagen I, lower viscosity is diluted to a lower concentration of 3mg/ml), thus making it is less viscous and much easier to handle.

Name

Catalog number

Pack size

Cultrex® rat collagen I, lower viscosity

3443-003-01

1ml @ 3mg/ml

3443-100-01

35ml @ 3mg/ml

Cultrex® 3-D collagen I (from rat tail tendons)

3447-020-01

20ml at 5mg/ml

The 5mg/ml rat collagen I, for example cat# 3447-020-01, is not treated with pepsin and therefore, it is highly viscous and has intact telopeptides. It is provided in acetic acid and needs to be pH neutralized before use. This material should never be pipetted with a narrow bore tip or a graduated 1ml pipette.

Instead, blue pipette tips (200-1000µL capacity) can be used on which sterile scissors are clipped to produce a wide bore. Users can consult the provided product data sheet for pH neutralization instructions. AMSBIO also offers 3mg/ml Cultrex® rat collagen I, lower viscosity (cat#s 3443-003-01 & 3443-100-01).

AMSBIO’s 3D collagen has been widely tested for its potential to promote growth and differentiation of cell types, visualized by morphology in 3D in vitro. However, laminin may need to be added as indicated in the product data sheet for MCF-10A cells.

Reagents

R-Spondin (RSPO1) expressing cell line

Roof plate-specific Spondin-1 (R-Spondin 1 or RSPO1) is a 27kDa secreted activator protein that is part of the R-Spondin family. It is also called CRISTIN3. Wnt/beta-catenin signaling is regulated by R-Spondins, probably by acting as an inhibitor and ligand for ZNRF3 and LGR4-6 receptors, respectively.

R-Spondin-1 performs the following functions:

  • Promotes proliferation of intestinal crypt epithelial cells
  • Supports renewal of intestinal epithelial stem cells
  • Increases healing of intestinal epithelial stem cells

The 293T cell line is steadily transfected to express murine RSPO1 with an N-terminal HA epitope tag and joined to a C-terminal murine IgG2a Fc fragment. Purified RSPO1 or RSPO1 conditioned media are then produced using this Fc 293T cell line. In organoid culture, the murine RSPO1 protein is widely used to preserve Lgr5+ stem cells, and the HA and FC tags make it easy to characterize or purify.

Figure 3. 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-Spondin1-Fc 293T cell line is cultured 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.

Benefits

  • Key medium component for most organoid culture models
  • Recombinant mouse RSPO1 protein is expressed by cell line
  • Wnt/beta-catenin signaling is positively regulated
  • Purified protein and conditioned medium from the cell line has been used for culturing human as well as mouse organoids

Name

Catalog number

Size

Cultrex® R-spondin1 (RSPO1) Cells 3

3710-001-01

1 vial (0.5ml), 1x106 cells

Proteins and antibodies

The Wnt family of secreted glycoproteins plays a key role in many critical cell functions such as cell migration, proliferation, polarity, self renewal, survival, and cell fate. Canonical Wnt signaling pathway has been extensively studied which regulates the amount of transcriptional co-activator β-catenin, which, in turn, regulates the expression of critical developmental genes.

Moreover, Wnt signals support organoid expansion. Wnt3a and RSPO1, which functions as an Lgr receptor agonist, are two major factors used for expanding organoids.

A series of RSPO1 and Wnt3a recombinant human and mouse proteins are provided by AMSBIO. The mouse proteins are expressed in CHO cells, while the human recombinant proteins are purified from HEK293 cells. Both proteins can be used for a range of cell assays and treatments.

R-Spondin (RSPO1) purified proteins

Name

Catalog number

Size

Human R-Spondin 1 / RSPO1 Protein

AMS.RS1-H4221-50UG

50μg

Human R-Spondin 1 / RSPO1 Protein

AMS.RS1-H4221-1MG

1mg

Mouse R-Spondin 1 / RSPO1 Protein

AMS.RS1-M5220-50UG

50μg

Mouse R-Spondin 1 / RSPO1 Protein

AMS.RS1-M5220-1MG

1mg

Wnt (Wnt3A) proteins

Name

Catalog number

Size

Human Recombinant Wnt3a (75% purity)

AMS.rhW1HEKL-001

1μg

Human Recombinant Wnt3a (75% purity)

AMS.rhW1HEKL-002

2μg

Human Recombinant Wnt3a (75% purity)

AMS.rhW1HEKL-010

10μg

Human Recombinant Wnt3a (85-90% purity)

AMS.rhW1HEKH-001

1μg

Human Recombinant Wnt3a (85-90% purity)

AMS.rhW1HEKH-002

2μg

Human Recombinant Wnt3a (85-90% purity)

AMS.rhW1HEKH-0010

10μg

Mouse Recombinant Wnt3a (75% purity)

AMS.rmW3aL-001

1μg

Mouse Recombinant Wnt3a (75% purity)

AMS.rmW3aL-002

2μg

Mouse Recombinant Wnt3a (75% purity)

AMS.rmW3aL-010

10μg

Mouse Recombinant Wnt3a (85-90% purity)

AMS.rmW3aH-001

1μg

Mouse Recombinant Wnt3a (85-90% purity)

AMS.rmW3aH-002

2μg

Mouse Recombinant Wnt3a (85-90% purity)

AMS.rmW3aH-010

10μg

Lgr5 (gastric stem cell marker) antibodies

Name

Species reactivity

Catalog number

Size

LGR5 mouse monoclonal antibody, clone UMAB212

Human

UM800104

100μl

LGR5 mouse monoclonal antibody, clone UMAB210

Human, Mouse

UM800102

100μl

LGR5 mouse monoclonal antibody, clone UMAB211

Human

UM800103

100μl

LGR5 mouse monoclonal antibody, clone OTI2A2

Human, Mouse

TA503316

100μl

LGR5 mouse monoclonal antibody, clone OTI7F2 (formerly 7F2)

Human, Mouse

TA808752

100μl

LGR5 mouse monoclonal antibody, clone OTI3F1 (formerly 3F1)

Human, Mouse

TA808748

100μl

Organoid harvesting solution

Cellular behaviors and morphologies exhibited by organoid cultures are similar to those viewed in vivo. However, the complexity in separating intact organoids from extracellular proteins containing the hydrogel has limited the adaptation of these models for investigating biochemical processes.

Usually, these extracellular proteins are degraded using proteases, but these proteases also tend to degrade proteins on the surface of the cells, and protease activity may also carry over into subsequent lysates or culture preparations.

A ready-to-use, non-enzymatic approach is provided by Trevigen’s Cultrex® Organoid Harvesting Solution for depolymerizing extracellular matrix proteins. This method enables harvesting intact organoids for cryopreservation, passaging, or biochemical analysis.

Benefits

  • Non-enzymatic chelating solution
  • Ready-to-use
  • Gentle for cells: preserves original morphology
  • Depolymerizes basement membrane matrix for harvesting organoids from culture

Applications

  • Sample preparation (PCR, Western blot, and immunohistochemistry)
  • Organoid passaging

Protocol

Figure 4. Protocol

Name

Catalog number

Size

Cultrex® Organoid Harvesting Solution

3700-100-01

100ml

Cryopreservation of Organoids

AMSBIO’s CELLBANKER® cryopreservation media ensures the stable long-term storage of cells and organoids. This series of easy-to-use cell freezing media offer high cell viability in serum and serum-free formulations to successfully cryopreserve the most sensitive and valuable mammalian cells. The latest formulation for stem cell freezing is GMP grade and comes with or without DMSO.

Advantages of CELLBANKER®:

  • Enables long term cell storage >10 years at -80°C or -196°C
  • Consistent high cell viability (> 90%)
  • Serum, serum-free and chemically defined formulations
  • Simple protocol – ready-to-use
  • Tested on many cell types

STEM-CELLBANKER® GMP, plus CELLBANKER® 1 and 2 have been shown to successfully cryopreserve liver organoids courtesy of Dr Meri Huch, Gurdon Institute Cambridge

References

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

  1. 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

Published protocols that have been used successfully with BME 2 RGF matrix for organoid growth

  1. 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).
  2. 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
  3. 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
  4. 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

About AMS BiotechnologyAMS Biotechnology

AMSBIO is a premier provider of quality life science research reagents and services helping customers develop innovative methods, processes, products and medicines. This is achieved by offering small and medium size manufacturers, academic groups and revenue generating biotechs a unique partnership for the global market and by providing state of the art and cost effective solutions to end users and partners.

Ambitious companies serving the global research science market need state of the art solutions to be able to generate success and help establish a critical mass. The current amsbio portfolio is a testimony to this. Specializing in Genomics, Proteomics, Cell Culture and Stem Cell Sciences.

AMSBIO continues to offer a wide range of solutions from leading manufacturing partners and academic technology transfer departments. Key areas include cell migration, invasion, adhesion and proliferation where a number of platforms suitable for high content analysis are available. Growing cells in 3D is physiologically relevant and the most innovative set of products and technology currently commercially available for 3-D cell culture has been put together under the AMSBIO umbrella. These products are being used in key regenerative medicine therapy and cancer research as well as offering alternatives to the use of animals in biomedical research.


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Last updated: Jul 13, 2017 at 6:11 AM

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