Despite a long history of using 2-D cell cultures in many areas of research, it is now well accepted that this model is not the best representation of a complex in vivo microenvironment. As such, 3-dimensional cell culture systems have emerged as pivotal models for many areas including drug screening, personalized medicine, bioengineering etc.
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1. What are 3-D cell cultures?
2. What is the advantage of 3-D culture over traditional 2-D culture?
3. What is a spheroid?
4. What is an organoid?
5. What is a Next Generation Organoid?
6. How should cells be cultured prior to setting up the 3-D culture?
7. What are the variables associated with 3-D culture in hydrogels of a biological origin?
8. Which matrix should I use for 3-D culture?
9. How do I handle hydrogels?
10. What are common methods of 3-D culture using biological hydrogels?
11. Can I transfer organoids from one matrix to another?
12. Can I use synthetic hydrogels to culture organoids?
13. I am working under GMP conditions. Is there an animal-free scaffold I can use to develop spheroids?
14. I am not very familiar with culturing on chips. How does this work compare to regular culture?
17. I am working on a multi-step long term experiment where I need to cryopreserve my organoids and then thaw them about a month later. What is the best way to do this?
18. What type of analysis is typically applied to 3-D cultures?
19. I am curious about scalability. For this to work for drug discovery, throughput must be fairly high. How can I increase my organoid throughput?
20. How does the cost of culturing organoids compare to 2D culture? What about time and lab resources?
3-D cultures are in vitro cultures where immortalized cell lines, primary cell lines, stem cells, or explants are placed within an environment that closely mimics in vivo conditions, thus enabling these cultures to develop into constructs with physiological functionalities similar to that seen in intact organisms.
There are multiple methods to achieve an environment that is suitable for development and maintenance of various 3-D cultures. This includes the use of:
While 2-D culture has been used for studying many aspects of cell function and behavior, the tissue-culture treated plastic environment is unlike anything found within living organisms. As result, cells in 2-D culture exhibit altered morphology, function, proliferation and gene expression when compared to their emanating tissues. By placing these cells in a 3-D environment, they assume biological and biochemical characteristics similar to what is observed in vivo thus yielding more accurate data.
Spheroids are simple clusters of broad-ranging cells, such as from tumor tissue, embryoid bodies, hepatocytes, nervous tissue, or mammary glands. They don't require a scaffolding to form 3-D constructs; they do so by simply sticking to each other. However, they can't self-assemble or regenerate, and thus aren't as advanced as organoids.
Organoids are derived from primary tissue, embryonic stem cells or induced pluripotent stem cells that retains the functionalities of the tissue of origin. Not only do they have the capability to self-assemble and self-organize into organ-like structures, they are also self-renewing.
They are complex clusters consisting of organ-specific cells, for example, the bowel, pancreas, liver. Within the 3D environment they grow into microscopic versions of parent organs viable for 3D study.
The Human Cancer Models Initiative (HCMI) started a project to create a range of Next Generation Cancer Models which include patient-derived organoids, conditionally reprogrammed cells, and neurospheres. HCMI uses current culturing techniques to develop such cancer models for use in translational cancer research as well as other applications.
Not only do primary patient-derived organoids retain the genomic and transcriptomic expressions of the primary tumors, they also have the benefit of long-term expansion in vitro enabling multiple analyses on the same sample material. The table below shows a more comprehensive comparison between various organoid techniques.
Refer to the product sheet included with your ATCC cell line for the sub-culturing procedure recommended for that particular cell line. Anchorage-dependent cell lines are usually sub-cultured by disaggregation of the cell sheet with proteolytic enzymes such as trypsin. Ethylene diamine tetra acetic acid (EDTA), a chelating agent, may be added to the dissociation solution to enhance the activity of the trypsin by removing calcium and magnesium from the surfaces of the cells. An appropriate solution for general use is a solution of 0.25% (w/v) trypsin to 0.03% (w/v) EDTA prepared in saline without divalent cations (such as calcium- and magnesium-free phosphate buffered saline).
Scaffolds used in 3-D cell culture plays multiple roles including providing physical support in the form of mechanical structures or ECM-like matrices. These methods usually involve embedding the cells into a matrix whereupon the physical and chemical characteristics of the matrix can influence cell behavior. Thus, it is important to carefully select the matrix to achieve a desired effect on the 3-D constructs.
The ECM is composed primarily of different glycosaminoglycans and fibrous proteins, such as collagen, laminin, and fibronectin. These components form an organized, complex network of locally secreted macromolecules that provide the structural framework for cell migration, adhesion, proliferation, and differentiation within the tissues of an organism. As such, different formulations of the ECM can be used to mimic the environment surrounding a particular tissue/organ.
The major variables associated using hydrogels for 3-D culture are cell type, cell seeding density, composition of hydrogel, thickness of hydrogel, stiffness of hydrogel, composition of cell culture medium, and time of culture.
Choice of matrix should correspond to the environment that you wish to recapitulate. A basement membrane extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Collagen I is the major constituent of connective tissue, and it is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.
When culturing organoids, the choice of BME used is crucial. The BME should provide high tensile strength, enhanced levels of entactin/nidogen, elevated protein concentration, and robust clarity and purity that ensures that the hydrogel contains the maximal amount of soluble extracellular matrix proteins.
Another point to consider is the batch-to-batch variability which stems from the hydrogel itself as it is sourced from natural materials, and as such might interfere with downstream analyses.
Most natural hydrogels in the market are viscous at lower temperatures (2-8°C) but polymerizes at temperatures above 15°C. As such, you should always handle thawed hydrogels on ice to prevent untimely gelling.
The two principal methods for conducting 3-D culture are the top assay and embedded assay. For the top assay, cells are seeded on a thick gel and a thin overlay is applied with the cell culture medium.
For the embedded assay, cells are resuspended within a thick gel and the culture media is applied on top. The top assay is easier to setup, to control seeding densities, and to keep cells within one focal plane for analysis.
Alternatively, you can resuspend your cells in the hydrogel and dispense onto culture plates in dome-shaped droplets. Once dispensed, invert your plate to help prevent your cells from settling onto the culture dish surface.
It is advisable to adhere to standard cell culture practice, where you would typically maintain the same matrix throughout your process of deriving and expanding your organoids. Generally, specific growth/differentiation factors are added to the matrix or media in lieu of transplanting mature organoids from one matrix to another.
The benefit of using synthetic hydrogels is that the formulation of the matrix is well defined and are often customisable to suit the desired level of stiffness. Common materials used to make up synthetic hydrogels are polyethylene glycol (PEG), polylactic acid (PA), and polyglycolic acid (PGA). While these materials are reproducible, they lack the biochemical components present in natural ECMs that influence the functionality of your organoids.
It has been shown that alginates and functionalized polyethylene glycol (PEG) formulations can support human pluripotent stem cell-derived intestinal organoids as well as organ progenitors from healthy mouse intestinal tissue. However, the hydrogels used had to be functionalized appropriately to provide a more optimized environment for organoid growth.
It is also important to note that many of these synthetic options may not be as robust as natural hydrogels in its ability to support organoid culture. For example, synthetic formulations may support organoid growth from healthy intestinal tissue but not patient-derived organoids; they may not support the various morphologies associated with organoid growth (budded vs. cystic forms).
Aside from the previously mentioned synthetic hydrogel, other xeno-free scaffold alternatives have also been used to develop 3-D constructs. As with the other hydrogels discussed above, a good matrix must be able to provide the appropriate mechanical and biochemical support seen in in vivo environments. Some common natural animal-free polymers are cellulose, alginate, and chitin. These polymers can be abundant, low-cost, consistent and tunable, thus increasing scalability potential for high throughput analyses.
Recent advancements in microfluidic technologies paved the way to developing organ-on-a-chip models. These chips are created using methods similar to computer microchip fabrication and then ‘printing’ living cells on these chips to create ‘organs’ with in vivo-like physiology.
The use of these microfluidic devices allows for better control over various parameters important for organoid culture such as extracellular matrix, fluid transportation, controlled medium exchange and spatial separation. These chips also permit high-resolution imaging in real time to enable better monitoring of the cultured organoids.
Below are a few examples of organoids cultured on a chip:
It is possible to culture 3-D cells without the use of a scaffold. These methods rely heavily on the self-aggregation of cells into constructs to recapitulate physiological characteristics of tissues. These cells will synthesize their own ECM, and thus allowing for natural cell-matrix interactions to occur. Size of spheroids grown using these approaches is dependent on the initial number of cells seeded but can grow to a size where oxygen and nutrient gradients similar to tissue can be observed.
If spheroids or organoids are grown under static conditions, the formed constructs do not experience shear stress. This is essential for avoiding modifying gene expression which would prevent them mimicking the in vivo tissue. However, the static conditions come with a price: a large depletion zone surrounding the spheroids/organoids. This depletion zone will starve (nutrients), poison (metabolic waste) and suffocate (O2) the cells, thereby reducing their longevity. The short lifetime will not provide the cells the time needed to recover structure, communication and especially O2 gradients which are essential for the spheroids/organoids to develop tissue-like functionality.
‘Active diffusion’ is needed to minimise the depletion zone. This should be obtained without creating shear stress. In a clinostat system, the spheroids/organoids stay in an almost static orbit, i.e. essentially not moving relative to the rotating bioreactor. The very gentle tumbling of the constructs in the media washes away most of the diffusion-depleted zone, increasing the steepness of the gradients – to the benefit of the constructs’ longevity.
There are two types of specialized microplates used to promote spheroid formation. Hanging drop plates have open, bottom-less wells designed to form small droplets of media. Cell suspensions – of either single or multiple cell types – are then added to the droplets and are allowed to self-aggregate over several days to form spheroids. They must then be transferred to a second vessel with higher media volume to ensure adequate nutrient supply for longer term cultures.
Similar to the hanging drop plates, low adhesion plates or ultra-low attachment (ULA) plates promote spheroid formation by preventing adherence of cells to the plates’ plastic surfaces. As the name suggests, these plates are normally treated with hydrophilic or hydrophobic coatings such as poly-HEMA or agarose. As the surfaces are not suitable for adhesion, cells will aggregate together to form spheroids.
Occasionally, magnetic levitation can also be used in conjunction with ULA plates to generate spheroids. This method involves treating cells with magnetic nanoparticles and are floated toward the air/liquid interface (ALI) of the well using a magnetic field.
Cryopreservation of organoids becomes more and more important to improve organoid-based therapy and for acquiring large numbers of cells. Organoids are typically cryopreserved at the same point in culture that they would otherwise be passaged. Organoids are cryopreserved intact, in fragments, or as dissociated cells.
The steps involved in initiating organoid cultures are similar to other cryopreserved cells. Cryovials are removed from liquid nitrogen storage and rapidly thawed. The contents of the vial are washed to remove the cryopreservation medium and generate a cell pellet. The pellet is re-suspended in liquid ECM and dispensed as small droplets onto tissue culture plastic. After a brief incubation at 37°C, the droplets solidify into a gel that can then be overlaid with warm liquid culture medium. Organoids grow and expand within these gel “domes.”
Full protocols for cryopreservation and thawing organoids can be found in the ATCC Organoid Culture Guide.
Within the cultures, cells may be assessed for morphology, apical/basal polarity, protein localization, and relative proliferation. Bioenergetics studies is also useful to determine the physiological state of the cultured organoids. For example, measuring the mitochondrial respiration and glycolysis provides a systems-level view of the cellular metabolic function of these 3-D constructs.
In addition, cells may be isolated from the 3-D culture and evaluated for levels of RNA and protein expression, as well as modifications to DNA. Furthermore, spatial profiling assays can be used to characterize tissue organization and the cytoarchitecture of the organoids.
The nature of organoids provides an excellent basis for drug discovery. As referenced in the question, a large volume of organoids would be required to move organoids into a high throughput screening environment. Although there are still currently challenges in this area, including labor-intensive work and time, there are protocols that briefly introduce how organoids can be expanded.
For scalability, organoids can be plated in single well plates. When preparing organoids for a drug screen, an extra split in which organoids are processed 1:1 and plated again for 1-2 days can increase the number of organoids significantly. This gives the organoids time to recover from the stress of disruptions and let them reach the appropriated size for screening activities (20-70 µm).
During the manipulation of the organoids for drug screening, it is recommended that you supplement the washing medium with RhoKi to reduce the stress that stems from the organoids not encapsulated by ECM. It is not recommended to store organoids on ice.
This question is particularly difficult to answer as there are no “standard” 2-D or 3-D cell culture protocols in which the format, volumes and protocols are comparable. In general, cell culture reagents/consumables used for 2-D are less advanced than those used for 3-D cell culture and therefore, the direct cost for 3-D cell culture are typically more than for an experiment with a conventional monolayer culture. Additionally, within a few days, a simple monolayer of cells can be formed; the generation of 3D cell structures like organoids might take up to several days, but during this period no elaborate steps need to be conducted by the cell culture personnel.
However, there are certain applications where conventional 2-D experiments will not lead to the same rich answers as experiments conducted in 3-D cell culture, as cells grown in 3-D more closely mimic in vivo behavior in tissues and organs. 3-D cell culture environments create more biologically relevant models for drug discovery which may lead to more predictive results, higher success rates for drug compound testing, a faster path to market, and reduced development costs.