Cell-based Assay

Andrei Leitão

A landmark study by Swinney and Anthony found that among the 183 small-molecule drugs across all

therapeutic areas approved between 1999 and 2008, 58 (32%) were discovered using phenotype-based approaches. Importantly, 28 (56%) of the 50 small-

molecule first-in-class new molecular entities (NMEs) identified in their study resulted from phenotypic screening approaches (phenotypic drug discovery;

PDD), whereas 17 (34%) resulted from target-based approaches.

Planning the assay

4 http://www.promegaconnections.com/considerations-for-successful-cell-based-assays-i-choosing-your-cells/

Type of cell

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Type of cell

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Primary tissue cell isolation

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Purification of primary cells

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Culture variables

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• pH

• Atmosphere (CO2 pressure)

• Buffer

• Medium: defined or undefined (with FBS, HS...)

• Use of chemical or physicial agents

• (Not) use of antibiotics and antifungals

• Contamination of cells

• Type of cell culture protocol suitable for the assay outcome

http://www.promegaconnections.com/considerations-for-successful-cell-based-assays-ii-cell-culture-conditions/

Freezing/thawing cells

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• Freezing: 1oC per minute

• Use of DMSO or glycerol

• Use FBS during freezing

• Thawing: fast

• Careful handling of the material

• Most critical step regarding contamination

Treatment parameters

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Timing

The optimal timing of treatment will be affected by the type of cell, the test compound, dosage and endpoint of the assay. For instance, if your assay measures caspase activity, you may need to look at earlier time points, because caspase activation is an early event in apoptosis. At later time points (24 to 48 hours) the cells may be dying through mechanisms of secondary necrosis, and assays for caspase activity would give falsely low results.

Drug Dose The drug dose affects the timing and synchrony of the cellular response, and it can even determine the type of response. For instance, some drugs that induce apoptosis at low doses may be acutely cytotoxic at higher doses.

http://www.promegaconnections.com/considerations-for-successful-cell-based-assays-3-treatment-parameters/

Assay technique

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• The assay technique should be suitable for the type of result you are looking for

• Critical analysis of the literature is important to identify bottlenecks and foresee/circunvent problems

• Delineate the conditions to perform the assay without struggling with equipment/structure

Controls

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• Essential for any type of assay

• Identification of the best compounds or biologicals is not that easy as found in the literature

• This comes to a topic named chemical (or biological) probe

• Nowadays na increased awareness is achieved to this question once many cases proved that chemical probes are misused

Arrowsmith, C.H. et al. The promise and peril of chemical probes. Nature Chemical Biology 2015, 11 , 536-541

Detection parameters

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Colorimetric studies depend on the wavelenght of detection nearby the peak of absorption

Fluorescence depends on the emission/excitation wavelenghts Problems may come from superposition of spectra when using a compound, leading to false-positive. The ratio signal-to-noise should be determined (Z and Z’)

3D cell culture

Andrei Leitão

3D models

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There are three main types of three-dimensional culture: (1) organ culture, in which whole organs, or representative parts, are

maintained as small fragments in culture and retain their intrinsic distribution, numerical and spatial, of participating cells;

(2) histotypic culture, in which propagated cells are grown alone to high density in a three-dimensional matrix or are allowed to form three-dimensional aggregates in suspension;

(3) organotypic culture, in which cells of different lineages are recombined in experimentally determined ratios and spatial relationships to recreate a component of the organ under study (Fig. 25.2)

R. Ian Freshney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th ed., Chapter 25

3D models

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2D versus 3D models

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200 µm Human hepatocyte cell – HepG2/C3A

Cell growth and viability

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200 µm

A,C,E: 2D culture B,D,F: 3D culture

Morphology

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A,B: 2D culture (4 days) C,D,R,F,G: 3D culture (21 days) BC: bile canaliculi-like structures G: glycogen granules N: nucleus M: mitochondria RER: rough endoplasmic reticulum SC: sinusoid-like channels packed with long micro TJ: tight junctions

Electron microscopy of C3A cells

5 µm

Urea and cholesterol production in immortal human hepatocytes

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A,C: 2D culture (4 days) B,D: 3D culture (21 days) Inhibition of cholesterol production by lovastatin

2D versus 3D models

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Resistance: 2D versus 3D models

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Bar graph of the cell viability at 96h after drug treatment for 2D A431.H9 monolayer culture and 7500-cell A431.H9 3D spheroid culture conditions.

Human epithelial carcinoma cell

3D models - Organoids

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Survival of cells in aggregates beyond about 250 μm in diameter (∼5000 cell diameters) starts to become limited by diffusion, and at or above 1.0 mm in diameter (∼2.5 × 105 cell diameters) central necrosis is often apparent. Culture at the gas-liquid interface is one approach to keep the gas exchange. Hyperbaric oxygen chamber can be used to increase its level.

Freshney, R.I. Culture of Animal Cells, 6th ed.

3D models - Organoids

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Small fragment of tissue on a filter laid on top of a stainless steel grid over the central well of an organ culture dish.

Freshney, R.I. Culture of Animal Cells, 6th ed.

3D models – Histotypic culture

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Histotypic culture is defined as high-density cell culture with the cell density approaching that of the tissue in vivo. Gel and Sponge Techniques Collagen gel Matrigel - composed of laminin, collagen, fibronectin, and proteoglycans

Hollow Fibers Spheroids Rotating Chamber Systems Immobilization of Living Cells in Alginate Filter Well Inserts Cultures of Neuronal Aggregates

3D models – Organotypic culture

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High density, three-dimensional culture involving the recombination of different cell lineages may be referred to as organotypic culture. The key event that distinguishes these constructs from histotypic culture is the introduction of heterotypic cell interaction (co-culture), including diffusible paracrine effects and signaling implicating the extracellular matrix.

Freshney, R.I. Culture of Animal Cells, 6th ed.

Scaffolds

and matrices

28 Freshney, R.I. Culture of Animal Cells, 6th ed.

Types of 3D culture

Scaffold-free spheroid system

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Tung, YC, Hsiao, AY, Allen, SG, Torisawa, Y, Ho, M, Takayama, S High throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst 2011, 136, 473-478

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Cell growth as a spheroid

Adrien Decheix - BIOalternatives, Inc: pancreas duct cell (PANC-1) spheroid within HA scaffold

Types of 3D culture

Rotating-wall bioreactor

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Advantage: high number of organoids in culture with higher size compared to other techniques. Major concerns: shear stress and mass balance.

https://www.ukessays.com/essays/biology/the-rotating-wall-vessel-bioreactor-biology-essay.php http://www.genengnews.com/gen-articles/rotating-bioreactors-for-manufacturing/1899/

Ovine lung

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Radtke, A.L., Herbst-Kralovetz, M.M.; Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models. J. Vis. Exp. (62), e3868

Rotating-wall bioreactor

Types of 3D culture Embedded-hydrogel technique

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2D 3D

Advantages • No damage or thin sectioning required to visualize whole intact tissue samples • Allows marking and visualization of long-range projections and subcellular

structures • Allows multiple rounds of molecular phenotyping • Applicable to multiple tissue types and sizes Disadvantages • Multiple-step process that takes place over several days/weeks • Immunostaining is time-consuming for thicker tissue samples • High start-up and consumable material costs

http://wiki.claritytechniques.org/index.php/CLARITY_Technique

Types of 3D culture Scaffold made by polymer

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3D scaffolds for in vitro laboratory applications are available in a variety of materials: metals, ceramics, polymers - natural and synthetic, and composites. Properties to consider include biocompatibility, wettability, mechanical properties, and surface chemistry.

Lee J, Cuddihy M and Kotov NA, Three-Dimensional Cell Culture Matrices: State of the Art. Tissue Engineering: Part B, 2008, 14, 61-86.

Types of 3D culture Scaffold made by (bio)polymer

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Alvetex® polystyrene polymer from AMSBIO with examples

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Protocol for the formation of human skin equivalents

David S. Hill et al. Mol Cancer Ther 2015;14:2665-2673

Human dermal fibroblasts: A: hematoxylin and eosin (H&E) in Media A for 18 days. B and C: H&E stained 35-day full-thickness human. D: electronmicrographs of a noncellularized Alvetex scaffold; E: 35-day full-thickness human skin equivalent; F: normal human skin. A–C, scale bars, 100 mm; D–F, scale bars, 75 mm; Epi, epidermis; Der, dermis.

Types of 3D culture Hollow fiber perfusion as scaffold

37 Freshney, R.I. Culture of Animal Cells, 6th ed.

Types of 3D culture Hollow fiber perfusion

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http://www.dddmag.com/articles/2010/04/simulating-vivo-drug-effects

http://www.pharmabioworld.com/features_john_cadwell.html

Types of 3D culture Filter Well Inserts as support

39 Freshney, R.I. Culture of Animal Cells, 6th ed.

Types of 3D culture Microchips

40 Huh D, Hamilton GA and Ingber DE, From 3D cell culture to organs-onchips. Trends in Cell Biology, 2011, 21, 745-754.

A micro-engineered liver-on-a-chip reconstitutes hepatic microarchitecture

Overview of 3D techniques

41 http://www.sigmaaldrich.com/technical-documents/articles/biology/3d-biomatrix-white-paper-3d-cell-culture-101.html

Option Pros Cons Research Stage

Scaffold-free systems

•No added materials •Consistent spheroid formation; control over size •Co-cultures possible •Transparent •HTS capable; compatible with liquid handling tools •Inexpensive

•No support or porosity •Limited flexibility •Size of spheroid limiting

•Basic research •Drug discovery •Personalized medicine

In vitro 3D scaffolds for laboratory applications

•Large variety of materials possible for desired properties •Customizable •Co-cultures possible •Medium cost

•Possible scaffold-to-scaffold variation •May not be transparent •Cell removal may be difficult •HTS options limited

•Basic research •Drug discovery •Cell expansion

Overview of 3D techniques

42 http://www.sigmaaldrich.com/technical-documents/articles/biology/3d-biomatrix-white-paper-3d-cell-culture-101.html

Option Pros Cons Research Stage

Gels

•Large variety of natural or synthetic materials •Customizable •Co-cultures possible •Inexpensive

•Gelling mechanism •Gel-to-gel variation and structural changes over time •Undefined constituents in natural gels •May not be transparent •HTS options limited

•Basic research •Drug discovery

Bioreactors •Several options available •High volume cell production •Customizable

•Cost •HTS options limited

•Basic research •Tissue engineering •Cell expansion

Microchips

•In vitro organ specific systems •High gas permeability •Transparent

•Commercial availability •Required expertise •Cost •HTS options limited

•Basic research •Drug discovery

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Factors to consider 3D cell culture options

http://www.sigmaaldrich.com/technical-documents/articles/biology/3d-biomatrix-

white-paper-3d-cell-culture-101.html