In Vitro

The term in vitro, literally "in glass", designates any experimental setup performed outside a whole living organism: in a test tube, culture plate, microplate well, microfluidic device, or on an isolated organoid. It is the foundation of preclinical research in biology, biochemistry, pharmacology, and toxicology.

In vitro experimental systems cover a wide spectrum. At the molecular scale, cell-free biochemical assays (kinetic enzymology, binding on purified receptors in solution, ITC, SPR, crystallography). At the cellular scale, immortalized lines (HEK293, HeLa, CHO, COS-7 for recombinant receptor screening), primary cells (hepatocytes, myoblasts, murine neurons, endothelial cells), and induced pluripotent stem cells (iPSCs) differentiated into cell types of interest. More recently, 3D cultures (spheroids, intestinal, hepatic, or cerebral organoids) and organs-on-chip (with microfluidic perfusion) bring in vitro closer to tissue complexity.

Advantages of in vitro are numerous: precise control of conditions (concentration, temperature, medium composition, absence of systemic metabolic interference), superior reproducibility, lower costs, high throughput (high-throughput screening in 384- or 1536-well plates), and ethics (alternative or reduction of animal use, following the 3R principle). Initial screening of peptide libraries (phage display, mRNA display, peptide arrays) is systematically in vitro.

Limitations are equally important to know. The absence of integrated physiology removes endocrine feedback loops, adaptive immunity, circulatory dynamics, real tissue distribution, and the blood-brain barrier. A peptide highly active in vitro may disappoint in vivo due to poor pharmacokinetics, rapid plasma degradation, lack of target barrier penetration, or unexpected hepatic/renal metabolism. Cell models can also diverge from primary phenotypes: artificial receptor overexpression, loss of transcriptional regulation, oncogenic transformations of immortalized lines that alter responses.

In the peptide research pipeline, in vitro plays several successive roles: identity and purity validation (HPLC, MS), target binding evaluation (SPR, fluorescence), cellular functional tests (cAMP, Ca²⁺, phosphorylation, proliferation), off-target selectivity profiling, metabolic stability evaluation (human liver microsomes, human/rodent plasma), permeability studies (Caco-2 for oral route, MDCK-MDR1 for blood-brain barrier), and finally cellular toxicity studies (cytotoxicity, cardiac hERG, in vitro Ames genotoxicity).

In vitro results are then triangulated with in vivo data in animal models and, where possible, ex vivo data on human tissues (biopsies, patient-derived organoids) to consolidate the preclinical profile before any clinical trial.