Cell Culture Final

What are the four phases of drug discovery and development?

• Drug discovery

• Preclinical development

• Clinical development

• Regulatory approval

What is the aim of preclinical studies?

Investigate the behavior of novel therapeutic agents in cell culture or animal models

Under which directive is animal research in the EU regulated?

Directive 2010/63/EU on the protection of animals used for scientific purposes

What is the final aim of Directive 2010/63/EU?

Replace all animal research with non-animal methods, such as:

• 3D cultures

• Computer simulations

What are some ways researchers discover new drugs?

• New insights into disease processes

• Testing molecular compounds for beneficial effects

• Identifying unanticipated effects of existing treatments

• Using new technologies to:

  • Target medical products to specific sites

  • Manipulate genetic material

What information do researchers gather after identifying a promising drug compound?

• Absorption, distribution, metabolism, and excretion

• Potential benefits and mechanisms of action

• Best method of administration (oral, injection, etc.)

• Side effects or toxicity

• Differences in effects across different populations

• Interactions with other drugs and treatments

• Effectiveness compared to similar drugs

What are the Good Laboratory Practice (GLP) regulations for preclinical laboratory studies?

Defined in 21 CFR Part 58.1: Good Laboratory Practice for Nonclinical Laboratory Studies

Where can exhaustive GLP guidelines for the EU be found?

Websites of the OECD and the European Commission

What EU directives are applicable for GLP regulations?

• Directive 2004/9/EC

• Directive 2004/10/EC

What is the definition of Good Laboratory Practice (GLP)?

A code of standards for testing medicines in laboratories during development

What are the key requirements set by GLP regulations?

• Study conduct

• Personnel

• Facilities and equipment

• Written protocols

• Operating procedures

• Study reports

• Quality assurance oversight

How large are preclinical studies typically?

Not very large but must provide detailed dosing and toxicity data

What happens after preclinical testing?

Researchers review findings and decide if the drug should be tested in humans

How long does it take for a drug to be approved from discovery?

About 12-15 years

What was the median cost of getting a new drug to market in 2020?

$985 million

What is the success rate of drugs in clinical trials?

• 30% pass Phase II-III

• 70% pass Phase III-V

Why do most drugs fail at Phase II and Phase III clinical stages?

• Poor efficacy

• Safety issues

What are the main reasons for high attenuation rates in drug discovery?

• Inappropriate preclinical testing methods

• In vitro models that do not sufficiently predict drug efficacy and safety

How can the success rate of drug development be improved?

Use of new technologies in preclinical testing and in vitro models to obtain more accurate data

Why are cell-based assays important in drug discovery?

Crucial for testing drug effects on cells

What is the most commonly used type of cell culture in drug discovery?

2D monolayers of cells grown on planar, rigid plastic surfaces optimized for cell attachment and growth

What advantages have 2D cultures provided over the decades?

Wealth of information on biological and disease processes

Why are 2D cultures still widely used despite their limitations?

• Simple and low-cost maintenance

• High viability of cultured cells during the culture period

• Extensive comparative literature available

What are the main limitations of 2D cultures?

• Do not replicate the complex tissue microenvironment

• Fail to maintain in vivo mechanisms of cell differentiation, proliferation, and function

What advancements have enabled the development of 3D culture models?

• Advances in cell biology

• Microfabrication techniques

• Tissue engineering

What are some types of 3D culture models?

• Spheroids

• Organoids

• Organs-on-chips

• 3D bioprinting

What should an ideal 3D culture model simulate?

• Tissue-specific microenvironment

• Cell-to-cell and cell-to-extracellular matrix (ECM) interactions

• Tissue-specific stiffness

• Oxygen, nutrient, and metabolic waste gradients

How are 3D cultures categorized?

• Non-scaffold-based systems

• Scaffold-based systems

What is the advantage of scaffold-based 3D models?

Better mimicry of cell-to-ECM interactions

What is the advantage of non-scaffold-based spheres of a certain size?

More amenable to cellular and physiological gradients

What are scaffolds made of?

• Biological origin materials

• Synthetic materials engineered to mimic ECM properties (stiffness, charge, adhesive moieties)

What factors should be considered when selecting a 3D cell culture scaffold?

• Physical properties:

  • Porosity

  • Stiffness

  • Stability in culture

• Biological properties:

  • Cell compatibility

  • Adhesiveness

What kind of tissues do hard polymers support in 3D cell culture?

• Skin

• Tendons

• Bone

What are patterned surface microplates used for?

Specific applications such as cell networking

What are examples of natural scaffolds?

• Fibrin

• Collagen

• Hyaluronic acid

What are examples of synthetic scaffolds?

• Polymers

• Titanium

• Bioactive glasses

• Peptides

How do scaffold-free systems work in 3D culture?

Rely on self-aggregation of cells in specialized culture plates

What types of culture plates promote spheroid formation?

• Hanging drop microplates

• Low-adhesion plates with ultra-low attachment coating

• Micropatterned plates for microfluidic cell culture

What are spheroids?

Simple, multicellular 3D models formed due to the natural tendency of adherent cells to aggregate

What types of spheroids can be generated?

• Tumor spheroids

• Embryoid bodies

• Hepatospheres

• Neurospheres

What gradients can develop within spheroids?

• Oxygen

• Nutrients

• Metabolites

• Soluble signals

What are the challenges in working with spheroids?

• Maintaining uniform spheroid size

• Forming spheroids from a small seed number of cells

• Controlling specific ratios of different cell types

• Lack of reliable, standardized, high-throughput compatible assays for drug screening

How do low-adhesion plates assist in spheroid cultures?

• Promote self-aggregation of cells into spheroids

• Have ultra-low attachment surface coatings to minimize cell adherence

• Possess well-defined geometry to position a single spheroid per well

What is the key advantage of using low-adhesion plates in spheroid culture?

• Ability to form, propagate, and assay spheroids within the same plate

• Enables high-throughput screening (HTS)

What are organoids?

• Dish-based, 3D developing tissues that show realistic microanatomy

• Also called organ buds

How are organoids classified?

• Tissue organoids

• Stem cell organoids

What defines an organoid?

• A collection of organ-specific cell types

• Develops from stem cells or organ progenitors

• Self-organizes through cell sorting and spatially restricted lineage commitment

• Mimics in vivo development

What happens in spontaneous neural differentiation in ES (embryonic stem) culture without inhibitors?

• Heterogeneous regions form containing:

  • Neural progenitors (SOX2, red)

  • Neurons (TUJ1, green)

What do microfluidic systems replicate?

• Specific fluid flow

• Constant temperature

• Fresh medium supply

• Flow pressure

• Chemical gradients similar to in vivo systems

What are the two main types of microfluidic systems?

• Single, perfused microfluidic chamber (one kind of cultured cells)

• Two or more channels connected by porous membranes (lined by different cell types, e.g., blood-brain barrier model)

What are microfluidic devices designed for?

• Cell cultures under perfusion

• Continuous supply of oxygen and nutrients

• Removal of metabolic waste

How can microfluidic devices mimic shear forces in vivo?

By exposing cells (e.g., endothelial cells) to blood flow-like conditions

What are the two types of barriers in microfluidic devices?

• Physical barrier (physically incorporated into the device)

• Non-physical barrier (supporting matrix mimicking ECM)

What applications do microfluidic devices allow?

• Continuous application of drugs or soluble molecules (e.g., growth factors)

• Fluid exchange between compartments with different cell types

What is the structure of the gut-on-a-chip?

• Two microfluidic channels separated by a porous flexible membrane

• Membrane coated with extracellular matrix

• Lined with human intestinal epithelial cells (Caco-2 cells)

What are the three modeled conditions of the gut microenvironment?

• Static culture in transwell plates

• Fluid flow on-chip at low shear

• Fluid flow on-chip with cyclic strain (mimics peristaltic motion)

How was the lung-on-a-chip designed?

• Human alveolar epithelial cells and pulmonary microvascular endothelial cells co-cultured on opposite sides of a stretchable porous membrane

• A vacuum applied to mimic lung tissue stretching during breathing

What integrated organ-level functions were reconstituted in the lung-on-a-chip?

• Inflammatory responses to intra-alveolar E. coli infections

• Endothelial recruitment of circulating neutrophils

• Neutrophil transmigration through the alveolar-capillary interface

• Bacterial phagocytosis

How was the lung-on-a-chip used to model human diseases?

• Pulmonary edema was recreated by administering interleukin-2 (IL-2) into the microvascular channel

• IL-2 caused fluid leakage into the alveolar compartment, mimicking IL-2-induced pulmonary edema in cancer patients

What is 3D bioprinting?

A process used to precisely dispense biomaterials to construct complex 3D functional tissues or artificial organs

What are the advantages of 3D bioprinting?

• Accurate control of cell distribution

• High-resolution cell deposition

• Scalability and cost-effectiveness

What are the characteristics of extrusion printing?

• Produces uninterrupted cylindrical lines

• Allows for a wider range of materials

• Causes reduced cell viability due to higher mechanical stresses on encapsulated cells

What is a key advantage of extrusion printing?

Parallel multi-bioink printing and tissue-vessel printing

What is the most common bioprinting technology?

Extrusion-based bioprinting is used in most commercial bioprinters

How does laser-assisted bioprinting work?

• Uses a laser as an energy source to deposit biomaterials onto a substrate

• Non-contact printing method → Ensures high cell viability

What are the advantages of laser-assisted bioprinting?

• High cell viability

• Suitable for highly viscous materials

What are the challenges of laser-assisted bioprinting?

• High equipment cost

• Unexplored parameters affecting droplet size and quality

• Unknown side effects of laser exposure on cells

• Complex control system limits widespread adoption

What are hydrogels?

• Highly hydrated hydrophilic polymer networks with pores and void spaces between polymers

• Absorb and retain large quantities of water

• Mimic ECM, making them commonly used in scaffolds

What are the types of hydrogels?

Natural Hydrogels (Collagen, Gelatin, Alginate, Fibrin, Hyaluronic Acid, Agarose, Chitosan, Laminin)

• Adhesive properties

• High cell viability

• Controlled proliferation & differentiation

Synthetic Hydrogels (Polyacrylic Acid, Polyethylene Glycol, Polyvinyl Alcohol, Polyglycolic Acid)

• Well-defined chemical, physical, and mechanical properties

• Customizable stiffness and porosity

What is the role of the ECM in biocompatibility?

• Must be degradable or integrable with natural ECM

• Should not generate harmful by-products

• Should not have negative interactions with cells

What are dECMs, and why are they important?

• Decellularized ECMs (dECMs) can be solubilized into bioinks for bioprinting

• More complex than simple bioinks → Closely resemble native tissue

What molecules make up the ECM?

✅ Matrix Proteins: Collagens, Elastin
✅ Glycoproteins: Fibronectin
✅ Glycosaminoglycans: Heparan Sulfate, Hyaluronan
✅ Proteoglycans: Perlecan, Syndecan
✅ Sequestered Growth Factors: TGF-β, VEGF, PDGF, HGF
✅ Secreted Proteins: Proteolytic Enzymes, Protease Inhibitors

How does ECM composition influence drug response?

• Enhances drug efficacy

• Alters drug mechanisms of action

• Promotes drug resistance

Why is vasculature important in bioprinting?

• Cells require proximity to a perfused microvasculature for nutrients & waste transport

• Without vasculature, cells in 3D engineered tissues die quickly

What happens to 3D engineered tissues without vasculature?

Necrotic regions develop within a few hundred microns of each cell

What is an example of vessel bioprinting?

• Carbohydrate glass lattice (green) fabricated via extrusion bioprinting

• Encapsulated in ECM (grey) containing live cells (yellow)

• Sacrificial lattice dissolves, revealing a perfusable vasculature (red)

What are the challenges of printing complex hollow structures?

• Use of sacrificial materials increases printing complexity

• Removal methods must be cytocompatible

Why is printing pre-vascularized tissues difficult?

• Lack of reliable methods for pre-vascularization

• Self-assembly of vascular features is too slow

What are the issues with bioink preparation?

Takes days to weeks due to cell culturing & biomaterial synthesis

What are the mechanical limitations of dECM bioinks?

• More biomimetic, but lacks mechanical strength

• Needs support from stronger but less bioactive inks (e.g., PCL)

What are the different types of cells used in 3D bioprinting?

Primary Cells

• Multiple cell types embedded in different hydrogels

• Requires many bioinks per print

Stem Cells

• Bioink formulations with growth factors & small molecules

• Guides site-specific differentiation

Clinical Applications

• Patient-specific cells to avoid immune rejection

What does ADME-Tox stand for?

Absorption, Distribution, Metabolism, Excretion, and Toxicity

What cell models are used to study drug absorption?

Caco-2 cells (human colon carcinoma) → mimic intestinal epithelium

What cell models are used to study drug metabolism & excretion?

• Hepatocytes (from donated livers)

• Immortalized hepatocyte cell lines (HepG2, HepaRG)

What cell models are used to study nephrotoxicity?

Human renal proximal tubule cells (primary & immortalized)

What is "Body-on-a-Chip" technology used for?

Preclinical drug testing (alternative to animal models)

Pharmacokinetics & pharmacodynamics modeling

Determining drug bioavailability & efficacy

How does Body-on-a-Chip simulate drug circulation?

• Gastric acid stomach chamber → simulates oral drug absorption

• Blood stimulant & dialysis membrane → models circulation & excretion

What are the scaling challenges in Body-on-a-Chip models?

• Imbalances in organ volumes

• Blood flow rates may not accurately reflect in vivo conditions

What are the key limitations of organ-on-a-chip models?

✅ Low culture volumes & cell numbers → Detection sensitivity issues
✅ ECM degradation over time → Low long-term cell survival
✅ Inconsistent cell seeding
✅ Microbial contamination risk
✅ Imbalance between complexity & practicality → Difficulties in obtaining high-resolution images

What challenges arise in obtaining accurate readouts?

• Readouts may fall above or below clinical endpoints

• Different measurement techniques may lead to inconsistent data

What are the key fabrication challenges?

Need for new materials → PDMS has inappropriate physicochemical properties for ECM mimicry

Why is developing sustainable cell sources important?

Reliable disease-specific cells are needed for accurate disease modeling

What technical improvements are needed for higher robustness (>1 month)?

✅ Better ECM stability
✅ Improved fluidic control
✅ Efficient bubble removal

What is a "universal blood substrate" challenge in Body-on-a-Chip models?

Standard cell culture media must be optimized for multiple cell types in a single system

What are primary cells?

• Cells taken directly from living tissue (e.g., biopsy material)

• Established for growth in vitro

• Exhibit normal physiology and closely represent the tissue of origin

Why are primary cells important in research?

Provide more relevant results than cell lines due to their authentic physiology

What is the lifespan of primary cells?

Have a limited lifespan; stop dividing (senesce) after a certain number of divisions, even under optimal conditions

What is a cell line?

• Formed after the first harvesting and subculture of a primary cell population

• Can undergo a certain number of subcultures

What is a continuous (immortalized) cell line?

• Population of cells with indefinite growth potential due to genetic transformation

• Can be cultured through a very high number of subcultures

• Immortalization may be spontaneous, viral, or chemically induced

What is a cell strain?

• A subpopulation of a cell line selected through cloning or other methods

• May undergo additional genetic changes

• Can become more or less tumorigenic than the parent line

• May be designated as a separate strain following transfection procedures