3D Cell Culture: Assay Guide

• Three-dimensional (3D) cell cultures are artificially-created environments in which cells are permitted to grow or interact with their surroundings in a 3D fashion.
• 3D cell culture more closely resembles physiological conditions and can provide a better indicator of success in vivo. Assays and kits exist which can help cultivate and quantify 3D cell cultures.

Background

  What is 3D Cell Culture?

  Pros and Cons

Cultivation

  Basement Membrane Matrix

  Alginate Hydrogel

Assays

  Harvesting Kit                             XTT

  3D viability assay                      WST-1

  LDH & Adenylate Kinase          WST-8

Of all the culturing models used today in drug discovery, none have shown more promise in the field of cancer research than three dimensional cell culture models. Especially in the study of cancerous cells, tumor spheroids composed of cell aggregates can be a remarkable tool to better assess the in vivo efficacy of interventions before they move to trials in non-human animals. A 2014 study on breast cancer is a powerful example of how such culturing techniques can model the heterogeneous tumour microenvironment.  

3D cell culture is the practice of cultivating cells across three dimensions, allowing for structures such as spheroids, in an environment that mimics the spatial usage seen in vivo. Unlike the practice of 2D cell culture, which usually involves growing cells in a monolayer upon plates, 3D cell culture facilitates growth by adding another axis for expansion and interaction.

In diseases such as cancer, re-creating the correct microenvironment is important for assessing the true efficacy of drugs and other intervention methods (Source). In a 2D culture environment, cells do not have the same communications with their surrounding neighbours, or with the extracellular matrix. In a 3D environment, these interactions can be readily modeled and studied more reliably.

3D cell cultures improve the function, differentiation, and viability of cells and better capture the microenvironment compared to conventional 2D cell cultures. 3D matrices provide a physiologically relevant screening platform, by mimicking in vivo responses, for many cell types including cancer and stem cells in developmental morphogenesis, pharmacology, drug metabolism and drug toxicity studies.

As displayed above, a major advantage in using 3D cell culture is that it is a better model for in vivo tissue environments and interactions.

To date, a major drawback to the usage of 3D cell culture has been a lack of reliable assays, and that the assays which did exist required destruction of the organoid or structure being studied. However, the drawbacks for 3D cell culture usage have been addressed with the production and usage of new, improved cultivation methods and assays. Similarly, the issue of low batch-to-batch variability has been corrected with additional features in scaffolds and matrices, and new studies showing the validation of these procedures (Source).

1) Basement Membrane Matrix

2) Alginate Hydrogel

The basement membrane is a fibrous extracellular matrix which serves as a boundary and as an adhesion platform in mammals. Our 3D Cell Culture Matrix BME Kit contains a basement membrane matrix and wash buffer to allow for the cultivation and measurement of 3D spheroid growth. Our 3D Cell Culture Ready-to-Use Scaffold Complete Kit contains an enzyme-free scaffold dissociation solution, neutralisation buffer and ready-to-use scaffolds in a 96-well plate form.

Alginate hydrogel can be used for the cultivation of plant cells in much the same way that the BME matrix can be used for mammals. Our 3D Cell Culture Matrix Alginate Hydrogel Kit (shown in the figure below) contains materials which can be used to create three dimensional spheroid growth for plant cells. It is also compatible with the 3D Cell Culture Ready-to-Use Scaffold Complete Kit.

Fig 1. Growth of HepG2 cells and MCF7 cells with no matrix vs. a matrix scaffold. Upon Calcein AM staining, cell viability was found to be unaffected even during long term matrix cultivation.

1. 3D Harvesting Kit
2. 3D Viability assay
3. Adenylate Kinase
4. Lactate Dehydrogenase
5. XTT
6. WST1
7. WST8 (coming soon)

Our 3D Cell Culture Non-Enzymatic Cell Harvesting Kit is an ideal solution for the isolation of cells and spheroids from matrices. Unlike trypsin and accutase— two commonly used proteases for matrix degradation—the gentle, saline-based solution used in this assay is ideal for cells which may be sensitive to protease digestion. This is an important factor if the cells being cultivated are to be assessed on cell-surface protein or signalling, as proteases have a higher potential to damage these processes and alter such physiological assessments. Our saline-based solution is optimised and standardised for the complete dissociation of matrices, especially those from our own cell culture kits.

The isolation of cells and spheroids from their matrix is a key step in the terminal assessment of cell passaging, biochemical processes, or protein analysis. Isolating these cells with a high viability rate for subsequent biochemical, protein and cell-based analysis is made possible with the use of this kit.

The 3D Cell Culture HTS Cell Viability Assay Kit assay uses gentle non-radioactive, non-enzymatics methods to test for viability in a sample. This assay kit provides a quick, user-friendly and high-throughput screening method for characterising and screening cell viability, cytotoxicity and apoptosis. Our kit provides a standardised fluorometric method for sensitive quantification of cells, which can detect as few as fifty viable cells in a well. It is suitable for assessing cell proliferation, cell viability, chemotaxis, cytotoxicity, and apoptosis. The kit contains a matrix dissociation saline solution, viability assay buffer, Calcein AM, and is compatible with all mammalian cell types.

Once the matrix and spheroid dissociate from the 3D culture, cell growth can be assessed. Similarly, the kit allows for the measurement of cell viability in response to growth factors, cytokines, mitogens and nutrients. The effects of cytotoxic or cytostatic compounds— such as anti-cancer drugs, toxic agents, or other pharmaceuticals which may influence cell growth or spheroid formation— can be measured.

Adenylate kinase is a ubiquitous protein which is present in the plasma membrane of all eukaryotic and prokaryotic cells. Upon damage to the membrane, this protein is released into the culture medium. Thus, AK is a powerful indicator of cytotoxicity, and can also be used as a proxy for the measurement of viability.

The GenieGlow Cytotoxicity Assay Kit is a quick and sensitive assay to measure cell viability in a three dimensional model when combined with 3D Cell Culture Non-Enzymatic Cell Harvesting Kit.

Lactate Dehydrogenase (LDH) is a stable enzyme which is quickly released into cell culture medium upon damage to the plasma membrane. It is present in all cell types and so is a strong indicator of cytotoxicity. A well-characterized method of assessing cytotoxicity in 3D cell culture is simply removing the media present and running the assay on a 96-well plate.

Our high-throughput LDH cytotoxicity assay is useful for estimating biomass, counting cells, and determining the health of cells and tissues. LDH is widely used for cytotoxicity testing in cultured cells as well as viability testing.

XTT cell proliferation assays use the water-soluble yellow tetrazolium salt XTT to detect cellular metabolic activity. During the assay, yellow XTT is reduced to a highly coloured formazan dye by dehydrogenase enzymes in metabolically active cells. Since this conversion occurs only in viable cells, the amount of the formazan produced is proportional to the number of viable cells in a sample. The formazan dye created in the assay is soluble in aqueous solutions and quantified by measuring the absorbance at a 450 nm wavelength using a spectrophotometer. Ultimately, the dye absorbance is proportional to the number of metabolically active, viable cells in each well. (Source)

Our XTT Cell Proliferation Assay Kit is optimised for high throughput assays, and requires no washing or other steps that could cause cell loss or variability. Our kit is not radioactive, and is ideal for the quantification of cell proliferation and viability in response to pharmaceutical, chemical, nutritional, and environmental interventions.

WST-1 is a compound used for the colorimetric assessment of cell viability. This reagent provides a simple and accurate method to measure cell proliferation, which is based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases.

Expansion in the number of viable cells results in an increase in the activity of the mitochondrial dehydrogenases, which leads to an increase in the amount of formazan dye formed. The formazan dye produced by viable cells can be quantified by measuring the absorbance at 440 nm. This method is non-radioactive, rapid and more sensitive than MTT, XTT, or MTS-based assays.

Currently, WST-1 is the most sensitive assay for viability available. Read more about our protocol on WST-1 Cell Proliferation Reagent here.   The assay can be performed in the same microtiter plate, and requires no extra steps ( washing, harvesting, cell solubilization, etc.).

WST-8 (also called CCK-8) is the newest, most evolved form of viability testing. It has recently been shown to be considerably less toxic than its predecessors MTT, XTT, and WST-1. (Full article here) In fact, as WST-8 is an assay which works extracellularly, it is functionally non-toxic. WST-8 tetrazolium salt is reduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. Formazan can then be measured with a spectrophotometer to determine the proportion of cells which are metabolically active, and thus viable.

After the assay is finished, the cells can be washed and reused in monolayer form.

Technical Note by Dr Elodie Alessandri-Gradt¹

Introduction

Airway infection is the 4th leading cause of death worldwide, primarily driven by viral infections. Apical-ut respiratory organoids are innovative 3D models that mimic the human respiratory epithelium². They can be used for various applications in the field of medical virology including analysing viral entry, virulence, cell pathogenesis, drug safety, immune response and even high-throughput drug screening studies.
However, due to the complex nature of organoids and 3D models, there remains a paucity of reagents capable of accurately measuring cell proliferation, viability, and cytotoxicity in these structures. In this study, we show that the Assay Genie 3D Organoid Cell Viability Assay is an excellent reagent to measure cell viability in 3D respiratory organoids in a high-throughput 384-well format and gives far superior results to other leading reagents.

Assay Principle

The Assay Genie 3D Organoid Cell Viability Assay is a highly sensitive, 1-step kit to quantify the number of live viable cells directly in cell culture supernatants and 3D Organoid models. This kit is an excellent choice for proliferation and cytotoxicity studies and been validated in 2D cell culture as well as Apical-Out airway organoids in a high-throughput 384-well format. This technology is based on the reduction of WST-8 Tetrazolium salt by mitochondrial dehydrogenases to an orange product which is directly proportional to the number of viable cells when measured at 450nm. WST-8 is highly soluble in cell culture medium with a low cytotoxicity profile and can be added directly to cultured cells with no pre-treatments or wash steps. It is extremely powerful for high-throughput and
sensitive experiments especially those requiring longer incubation steps such as 24 hour or 48 hours. This kit offers a much higher sensitivity and lower cytotoxicity than other Tetrazolium reagents such as MTS, MTT, PrestoBlue or XTT.

Materials & Methods

Multimode reader
384-w dark plate with clear bottom Corning®
DMSO (Sigma)

Protocol

- 75 to 100/well home-made apical out organoids were dispensed into a 384w plate in triplicate
- 0.1% and 10% DMSO were added as negative and positive controls in a final volume of 50μl
- The plate was incubated for 2 days at 37°C with 5% CO2
- 5μl of 3D Organoid Cell Viability Assay CCK8 buffer or Prestoblue reagent was added to each well and
incubated for either 1 hour, 3 hours or overnight
- The absorbance of the wells containing the 3D Organoid Cell Viability Assay CCK-8 buffer was
measured at 490nm while those containing PrestoBlue reagent at ex 535nm/em 595nm
- Cell viability (%) was calculated from OD sample, control and blank.  

Results & Discussions

Table 1: Datasheet obtained with Spectra.

The 3D Organoid Cell Viability Assay gave excellent results for apical out airway 3D organoids cell viability, by comparison to another resazurin-based method.