How do organoids differ from primary 3D cell cultures? (2023)

How do organoids differ from primary 3D cell cultures? (1)

Published on January 16, 2020VonRajendra Kumari, Dr.

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Learn the key differences between organoids and 3D cultured primary cells from healthy tissue and tumors.


How do organoids differ from primary 3D cell cultures? (3)

in vitroMethods for establishing 3D cultures

in vitroSystems are essential tools for basic research, drug discovery and development. While conventional 2D cell culture methods were used in the past, advanced 3D methods are now being usedin vitroCultural systems are becoming more and more widespread.

While 2D cultures are propagated as flat monolayers on plastic, 3D cultures are propagated on plasticin vitroCulture enables complex spatial interactions between cells in a 3D environment. This is a more detailed summaryDirectCell-cell contacts and primary physiological conditions. This in turn provides a more appropriate microenvironment and influences cellular processes and therapeutic response. 3D breeding is also important to increase the establishment successin vitroModel system using fresh primary cancer cells.

(Video) Introduction to 3D Biology: Organoids, Spheroids and Applications Relative to 2D Culture

There are many 3Din vitroavailable model options using cell lines or tissues from the clinic. Due to the inability to translate 2D cell lines, tissue from a clinical source is the gold standard and is used in a variety of ways to develop model research systems. This also includes the complex generationorganoidityand 3D primitive cultures.

Because both models are generated directly from patient tissue (including tumors), they better reflect the original genetic makeup and gene expression profile of the tissue. However, there are some important differences between organoids and 3D primary cells that affect typical applications for each model type and are discussed below.

Main features of organoids

We previously discussed how organoids are normally formedadult stem cells (ASC) or embryonic stem cells (ESC)in the organ and how this process was perfected by Hubrecht Organoid Technology (HUB).both healthy and diseased tissue, such as cancer.

Briefly, organoids are:

  • self-organizing"This is an essential feature of organoids, from which multicellular structures arise."stem or stem cellsand shows striking similarities withDirectorgan architecture. Organoids are physiologically very important and contain many different cell lineages that interact and establish cell-cell contacts and replicateDirectprocesses.
  • Genomic stable"Because the organoids are derived from stem cells and stored under specific culture conditions, they both recapitulate the original genomic composition of the tissue and demonstrate genomic stability across multiple passages."
  • self-renewing"This means that organoids can be propagated and cryopreserved to form biobanks and resuscitated for genetic testing without compromising genetic identity."

Using HUB technology, both organoids and tumor organoids can be easily created, expanded, assembled and scaled for drug development applications, including large-scale drug screening.


Tumor organoids are developed from a variety of sources - clinical patient specimens, patient-derived xenograft models (PDX cultured in mice), and tumor tissues from mice. Tumor organoids derived from PDX are namedgeduldiges Xenograft-Organoider (PDXO)and represent an alternative ASC source for the patient's tissue, which may limit model development.

Tumor organoids developed from patient tumors and PDX retain primary tumor morphology and genetic properties such as gene amplification, somatic copy number, and mutations.

It is important that the tumor organoids that are important for the patient are also importantRepeat the patient's answer in the clinic, which ensures high translatability for all your applications.

Additional advantages of PDXO include the rapid development of large panel models of multiple tumor types, subtypes and mutations from the wide range of PDX collections already available. These PDXO panels capture the heterogeneity and diversity of the clinical cancer population that is lacking in the conventional cancer populationin vitroCell Line Panels.

Main features of primary 3D cell cultures

3D primary cell cultures utilize cells obtained directly from tissue and grown in scaffold-based or scaffold-free systems. Cultures are established from a single cell type or a multicellular mixture from a single tissue or different tissue types, including diseased tissue or tumor tissue.

Primary Cancer Cells

Primary cancer cells are expected to be much closer to the original tissue than traditional cell lines, and their main characteristics are:

  • Primary cells were not perpetuated or adapted for growthin vitroTherefore, the original genomic and expression profiles of the tumor are more accurately recapitulated.
  • In 3D, primary cancer cells reproduce the architecture of parental cancer tissue and more faithfully reproduce cellular processes and responses to therapy than 2D alternatives.

Tumor-derived primary cells can also be cultured in scaffold-free systems to form free-floating spheres called "Cancer spheresUnlike other types of 3D-cultured primary cell aggregates that contain more differentiated cells, tumor spheres are enriched with cells with cancer stem cell (CSC) characteristics.

Other types of assays using primary cells include methylcellulose clonogenicity, soft agar clonogenicity, and three-dimensional tumor growth assays (3D-TGAs), all for specific purposes. e.g3D-TGAexamines the role of the tumor microenvironment (TME) in drug response. Here, tumor cells are embedded in an extracellular matrix with low rigidity (IrBME, Cultrex®) and cultured together with hMSCs (e.g. IL-6, HGF) or CAF in order to mimic paracrine signaling in the TME of solid tumors.

Differences between organoids and primary 3D cell cultures

Although both organoids and 3D primary cell cultures can be derived from patients, there are important differences between the model types.

cultural conditions

Organoids are derived from stem cells and grown in very specific culture media to ensure thisproper growth and development of organoids. This is achieved by optimizing the growth conditions of the culture, for example by providing a basement membrane matrix (i.e®) and addition of selected agonists (e.g. Wnt and receptor tyrosine kinase) and inhibitors (e.g. bone morphogenetic protein/transforming growth factor-β).

The HUB team fine-tuned these growth conditions to enable the development of tumor organoids from cancer stem cells across the spectrum of cancer types. For better control of experimental data, organoids can also be obtained from matched healthy tissue.

(Video) 3D Cell Culture

Organoids developed with HUB protocols are epithelial in origin. On the other hand, primary cell cultures consist mainly of differentiated cells, and culture conditions can vary from culture to culture, making it difficult to establish a robust culture in certain cancers.


Organoids are based on complex processes of self-organization that lead to "mini-organs". Instead, primary cells, through simple cell-cell adhesion, produce multicellular structures that resemble spheroids rather than organ-like structures when a physical or mechanical force (e.g., excitation) is applied.

Long term maintenance

long term,in vitroCell expansion in culture requires a population of immature stem cells to replenish dying cells. This is because organoids originate from and maintain the stem cell populationin vitroculture, this guarantees their long-term viability. Organoids also preserve cellular composition, architecture, and genetic profiles across multiple passages.

In contrast, typical primary cell culture conditions are not optimized for long-term maintenance of the culturein vitroand cells tend to age or drift after a few passages.


The organoids have been successfully frozen and can be stored as isliving biobanks Ex-alivewithout affecting their genomic and morphological identity. This allows the use of biobanks for repeated research.

In contrast, primary cells are more of a disposable solution. While primary cells can be frozen for use in replicate studies, they are more difficult to revive effectively. Therefore, in repeat studies, returning to the original tumor tissue to collect the cells again is required.

Complex study

Because of their key features discussed above, tumor organoids can be enlarged on a large scalein vitroScreening of drugs that have a greater clinical relevance than todayin vitroHTS platforms. Large-scale screening of multiple drug candidates and combinations simultaneously saves time, effort, and money compared toDirectscreens.

With the help of primary tumor cells, the effectiveness of cancer drugs can be evaluated in simple 3D test systems. As with organoids, cells can be obtained directly from a patient's tumors orPDX-Modellierer. If PDX is used, cells are stripped of any mouse stromal components prior to use.

AppropriateDirectrole models

pairingin vitroOrganoid models with matchingDirectModels (e.g. PDXO and Matched PDX) provide a platform for better reliefDirectModel selection and enables the changein vitrofor in vivo studies using a customized platform that is highly relevant to the patient.

With suitable primary cell cultureDirectModels not always available, primary cell discoveries in progressDirectThe research requires additional steps to identify suitable models.


Both organoids and primary 3D cell cultures are superior to traditional 2D monolayer cultures because they provide a more physiologically appropriate environment for cell-cell interactions and cellular responses. Both have been successfully applied for clinical tissue usein vitroModel systems and are increasingly being used, particularly in anticancer drug development and discovery. However, there are some important differences between organoids and primary 3D cell cultures that need to be understood in order to select optimal research applications.

3D primary cell culture systems can be summed up betterDirectas 2D monolayer cultures and provides a simple and user-friendly platform for evaluating the efficacy of new drug candidates.

In comparison, however, organoids derived from stem cells offer additional advantages. These include the physiological importance of the primary organ and susceptibility to long-termin vitroCultivation of both healthy and diseased tissues to obtain heterogeneous stem cell cultures and differentiated progeny, expansion to viable tissue biobanks, HTS and more accurate prediction of patient response.

The use of organoids in drug development embodies the patient throughout the laboratory conceptin vitroIDirectand accelerate decision-making in the right direction with clinical utility.

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Rajendra Kumari, Dr.

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Kumari, R. (2020)How do organoids differ from primary 3D cell cultures?- The crown of life sciences.

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How do organoids differ from primary 3D cell cultures? ›

Organoids rely on complex self-organization processes resulting in “mini organs”. Primary cells, instead, generate multicellular structures via simple cell-cell adhesion when a physical or mechanical force is applied (for example agitation), resembling spheroids rather than organ-like structures.

What is the difference between organoids and 3D cell culture? ›

Length of time 3D cultures can be maintained: Long term, in vitro expansion of cells in culture needs an immature stem cell population to replenish dying cells. Organoids are derived from, and maintain, a population of stem cells during in vitro culture, which guarantees their long term viability.

What is the difference between organoids and cell cultures? ›

Compared with immortalized cell lines, organoids are considered superior in recapitulating the 3D architecture, heterogeneity, and cell functions of the primary tissues and hence, are more physiologically relevant for modeling human diseases and predicting drug response.

What is the difference between cell culture and primary cell culture? ›

Posted July 22, 2020. Primary cell culture is the culture of cells directly isolated from parental tissue of interest; whereas cell line is the culture of cells originated from a primary cell culture, which is generally used to expand cell population and prolong life span.

What are the advantages of organoids over two dimensional cell culture and animal models? ›

Given their self-renewal ability organoids can be cultured for longer periods of time. Organoids have a number of benefits including their recapitulation of human physiology, quick establishment and robustness compared to animal models, genetic modification ability, and patient-specific applications.

Why is 3D cell culture better? ›

Here are some reasons to use 3D cell cultures:

3D cultures better mimic tissue-like structures. Able to exhibit differentiated cellular function. Possible to co-culture two or more different cell types. Can simulate microenvironment conditions such as hypoxia and nutrient gradients.

What are the advantages of 3D cell culture? ›

3D plate cultures also show a higher degree of structural complexity and retain a “steady state” (homeostasis) for longer. Interaction between different types of cells — Creation of complex systems linked together by microfluidics means that 3D tissue systems can better model how different types of cells interact.

What are the different types of cell culture primary cell culture? ›

The most popular types of primary cells used in research are epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic and mesenchymal stem cells.

What are the advantages of primary cell culture? ›

Other than being an outstanding model for studying cell physiology and biochemistry, primary cell culture has various applications in vaccine production, drug discovery, and gene therapy. Primary cell cultures also reduce the burden on animal models and make research cost-effective.

What is the difference between primary and secondary cultures? ›

The main difference between primary and secondary cell culture is that the primary cell culture contains the cells directly obtained from host tissue, whereas the secondary cell culture contains sub-cultured cells from primary cell culture.

What are the limitations of 3D organoids? ›

Despite their numerous benefits, organoids have certain limitations, including a lack of vascularization and immune cells and the absence of an air-liquid interface due to their cystic form (2, 210) .

Why are organoids better? ›

In these artificial conditions, stem cells cannot only proliferate but also self-organize into complex structures. Compared with traditional 2D culture systems, 3D organoids better resemble the native organ in terms of gene and protein expression, metabolic function and microscale tissue architecture.

What are the pros and cons of using organoids? ›

Organoids offer superior morphology if you are studying a glandular tissue, but they are not appropriate for studying stratified tissues, such as skin. Originally organoid cultures were grown to investigate normal cellular differentiation in the prostate3 and breast.

What are the limitations of organoids? ›

Lack of a vascular system

The human body is dynamic; blood constantly flows through the blood vessels to exchange nutrients, oxygen, and waste ensuring cell survival. However, organoids lack a vascular system and are cultured through static methods.

What are the characteristics of organoids? ›

Organoids are tiny, self-organized three-dimensional tissue cultures that are derived from stem cells. Such cultures can be crafted to replicate much of the complexity of an organ, or to express selected aspects of it like producing only certain types of cells.

What is 3D organoid culture? ›

Organoids are in-vitro derived 3D cell aggregates derived from primary tissue or stem cells that are capable of self-renewal, self-organization and exhibit organ functionality.

What are the limitations of 3D cell culture? ›

However, it does have a few disadvantages. The major disadvantage of 3D cultures is that they are labor intensive and time consuming. 3D culture requires the separation of individual cells from spheroid structures by proteolytic degradation of single layers.

What are the problems with 3D cell culture? ›

Basic issues include the inability to scale a single 3D format up or down and the poor reproducibility between batches of biomimetic scaffolds. Although conventional cell culture techniques are still widely used by researchers, microfluidic systems are proving useful tools.

What is the main benefit of using a 3D model to study a cell instead of a 2d model? ›

The main advantages of 3D cell culture include more in vivo-like cell interactions, cell division, and morphology, as the 3D shape more accurately mimics the natural environment of cells.

What are two advantages of making 3D models? ›

Not only does 3D modeling help the designers and end users visualize space requirements, but also improves drawing efficiency and accuracy. 3D modeling for design allows the designer to see what they would not see when designing in 2D.

What are the advantages and disadvantages of 3D Modelling? ›

Pros and cons of these models

That is, models will always transmit to the visualizer, the objective form of their representation, without any place to interpretations or subjectivities. The cons about 3D models are that more and more models and mockups are being replaced in real life.

What is the advantage of 3D models? ›

Design Competence – Simply expressed, 3D modeling aids in the reduction of design time and costs. Before going into production, 3D CAD software allows each component of a structure or product to be reviewed, tested, and updated. This keeps you from having to go back to the drawing board.

What are the 3 types of cell culture? ›

Cells cultured in the lab can be classified into three different types: primary cells, transformed cells, and self-renewing cells.

What are the advantages and disadvantages of primary cells? ›

Advantages & Disadvantages of these type of batteries:

Low current (low C-rate) only. Even high current types are considered low in comparison to rechargeable batteries. Less environment friendly than rechargeable batteries. For large batteries – usually not cost effective.

What are the different methods of primary culture? ›

A primary culture is that stage of the culture after isolation of the cells but before the first subculture. There are four stages to consider: (1) acquisition of the sample, (2) isolation of the tissue, (3) dissection and/or disaggregation, and (4) culture after seeding into the culture vessel.

What are the challenges of primary cell culture? ›

Primary cells, however, are notoriously difficult to transfect and the efficiency varies greatly between different cell types. In addition, primary cells have a finite lifespan and limited expansion capacity, making it difficult to obtain a high yield of RNA.

What are two disadvantages of primary cells over secondary cells? ›

Primary cells v/s secondary cells
S.NoPrimary cellSecondary Cell
3Cannot be rechargedCan be recharged
4Internal resistance is high.Internal resistance is low.
5Can supply weak currents only.Can supply weak and high currents.
6Light and cheap.Heavy and costly.
3 more rows

What is the disadvantage of primary cell over secondary cell? ›

The only disadvantage of the Primary Cell is that it can only have a limited amount of oxidising and reducing agent which is why it cannot be charged for a long time. But in Secondary Cell Energy can be stored as fuel for longer period of time.

What is the characteristic of primary culture? ›

Primary cell culture: cell lines directly expanded from tissues. Unless they undergo an immortalization procedure, primary cells have a limited lifespan and usually reach senescence after 10–20 passages.

What is the key difference between a primary cell culture and a secondary cell culture quizlet? ›

-A primary culture is when cells are obtained directly from an organism. -A secondary culture is derived from a primary culture into long-term growing cells.

What do you mean by primary cell culture? ›

Primary cell cultures are defined as those growing from tissue explants or from individualized cells obtained from the original tissue. Their phenotype is closely related to that of the cells making the tissue. As such, primary culture is the gold standard for renal function studies.

What does 3D organoid culture support differentiation of? ›

3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR T cells. Cell Stem Cell.

Are all organoids 3D? ›

Organoids are three-dimensional (3D) cell structures grown in vitro from stem cells, primarily isolated either from biopsies or from pluripotent stem cells, that recapitulate key features of both the development and performance of native organs [1].

What are the potential uses of organoids? ›

Organoids may be employed as a source of intestinal regenerative medicine by developing the organoid within a complex three-dimensional scaffold to supply functional tissues. Intestinal organoids may also be transplanted back into the body to undergo functional maturation.

What are the challenges of organoids? ›

Despite the promising features of organoids, their broad utility is tempered by a variety of limitations yet to be overcome, including lack of high-fidelity cell types, limited maturation, atypical physiology, and lack of arealization, features that may limit their reliability for certain applications.

What are the benefits of 3D organoids? ›

Organoids are 3D in vitro cell cultures that may better recapitulate disease heterogeneity and retain parental tumor characteristics. Short-term ex vivo culture of PCa tissues may also facilitate drug testing in personalized medicine.

Why are organoids better than animal models? ›

Using organoids instead of animals is also cheaper and faster. And they can reveal much more about the inner workings of exceedingly complex organs, like the brain. So far, brain organoids are mostly being developed to study brain disorders, potential treatments for a specific patient, and toxic compound exposures.

What are the ethical issues of organoid research? ›

Research on and use of cerebral organoids raise a host of ethical issues ranging from traditional research ethics questions, including informed consent, biobanking, and data protection, to issues of clinical translation concerning, for example, the uncertain evidence basis for first-in-human trials or the risk of ...

What are the ethical implications of organoids? ›

Ethical considerations of organoid models. Organoids are likely to affect policies for research using animals and human embryos. They also have implications for biobanking and patient consent policies and require particular responsibility in communicating results to the public.

Can organoids replace organs? ›

Although patient-derived organoids exhibit substantial variability, they could eventually become useful for personalized medicine applications [9, 26]. A major prospect for organoids is their potential use as transplantable organs (Fig. ​ 2A).

Are organoids 2D or 3D? ›

Organoids are in-vitro derived 3D cell aggregates derived from primary tissue or stem cells that are capable of self-renewal, self-organization and exhibit organ functionality.

What is organoids cell culture? ›

Organoids are tiny, self-organized three-dimensional tissue cultures that are derived from stem cells. Such cultures can be crafted to replicate much of the complexity of an organ, or to express selected aspects of it like producing only certain types of cells.

What is the difference between 2D and 3D cell culture? ›

In 2D cell cultures, cells are grown in a flat plane on top of a flat surface, whereas in 3D cell culture they are grown in a three-dimensional space, usually embedded within a gel-like matrix, or grown in a solid scaffold.

What are the advantages of 3D cell culture over 2D cell culture? ›

A study showed that HepG2 liver cells cultured using a 3D polymeric hard scaffold were less affected by cytotoxic compounds and had greater viability than those grown in 2D. Furthermore, polymeric hard scaffolds are extremely useful in studying tissue regeneration as well as testing tumor cell treatments.

What are the disadvantages of 3D cell culture? ›

The major disadvantage of 3D cultures is that they are labor intensive and time consuming. 3D culture requires the separation of individual cells from spheroid structures by proteolytic degradation of single layers. This can take anywhere from several hours to a few days.

What are organoids as 3D model for? ›

Brain organoids are 3D tissue models representing one or more regions of the brain. They can overcome the shortcomings of conventional post-mortem and animal brain models to produce clinically relevant results. Cerebral organoids have great potential for understanding brain development and neuronal diseases.

What are the basics of organoids? ›

Introduction. Organoids are primary patient-derived micro tissues grown within a three dimensional extracellular matrix that better represents in vivo physiology and genetic diversity than existing two-dimensional cell lines.

What are organoids in simple terms? ›

A 3-dimensional, mini-organ-like structure made by growing a person's tumor cells or stem cells (cells from which other types of cells develop) in the laboratory. Organoids contain many types of cells and closely mimic the structure, organization, and some of the functions of human tissues and organs.

What is one huge difference between 2D and 3D design? ›

In 2D and 3D, the "D" defines the dimensions applied in the shape. The main difference between these shapes is that a 2D shape contains two dimensions, including length and width. In contrast, a 3D shape contains three dimensions, including length, width, and height.

What is a 3D cell culture model? ›

What is 3D Cell Culture? 3D cell culture is a culture environment that allows cells to grow and interact with surrounding extracellular framework in three dimensions. This is in contrast with traditional 2D cell cultures in which cells are grown in a flat monolayer on a plate.

What is the main difference between 2D and 3D? ›

A 2D shape is a flat object. It has length and width but no height (ex: square, circle, etc.) and a 3D shape is a shape in space. It has length, width and height (ex: cube, sphere, etc.).


1. Organoids: Cancer in 3D - Sanger Institute
(Wellcome Sanger Institute)
2. Growing Organs | Stem cells, Organoids and 3D Bioprinting.
3. Current Trends in 3D and Organoid Cell Culture for Cancer Research
4. Advances in Three-Dimensional Cell Culture in Drug Research and Discovery
5. 3D Organoid Culture Systems Infection Study by Microinjection | Protocol Preview
(JoVE (Journal of Visualized Experiments))
6. How to Culture Mouse Intestinal Organoids: Isolating Intestinal Crypts and Establishing Organoids
(STEMCELL Technologies)


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