Ex vivo

Ex vivo (Latin for 'out of the living') refers to biological studies involving tissues, organs, or cells maintained outside their native organism under controlled laboratory conditions. By regulating temperature, oxygenation, nutrient delivery, and—when whole organs are used—actively perfusing a nutrient-rich medium through the tissue's vasculature, researchers can sustain function long enough to conduct experiments that would be impractical or unethical in an intact organism. Positioned between in vitro (lit. 'within the glass') and in vivo (lit. 'within the living') studies, ex vivo models combine a relatively high degree of experimental control with substantial physiological relevance.

Ex vivo brainstem: A. coronal view displaying the anterior portion of the tissue sample; B. sagittal view displaying the left-hand side of the tissue sample^[1]

These platforms support pharmacologic screening, toxicology testing, transplant evaluation, developmental biology, and investigations of disease-mechanism research across medicine and biology, from cardiology and neuroscience to dermatology and orthopedics. Because they frequently employ donor tissue or surgical waste, they can reduce reliance on live-animal experimentation; their utility, however, is limited by finite viability, incomplete systemic integration, and post-mortem biochemical changes that accumulate over time. The earliest perfusion studies were conducted in the mid-19th century, and subsequent advances in sterilization, imaging, and microfluidics have facilitated broader adoption into the 20th and 21st centuries. Regulatory oversight depends on specimen origin: human ex vivo research is subject to informed consent and biobanking laws, whereas animal-derived models fall under institutional animal care guidelines.

Principles and definition

Ex vivo, meaning 'out of the living' in Latin, refers to biological studies involving tissues, organs, or cells maintained outside their native organism under tightly controlled laboratory conditions. These studies preserve the extracted materials' functional viability and structural integrity for limited periods by precisely managing conditions such as oxygenation, temperature, nutrient delivery, and humidity, depending on the requirements of the model. The conditions are often facilitated through cell culture media or specialized perfusion chambers.^[2][3][4] As an intermediate approach between in vitro studies—which typically use isolated cells in artificial environments—and in vivo research within living organisms, ex vivo models preserve more of the native tissue architecture than traditional cell cultures, while offering greater experimental control than whole-organism studies.^[5][6][7] By doing so, they address some limitations of in vitro work—such as oversimplified cellular interactions—and reduce the variability and systemic complexity of in vivo models.^[8][9] In vitro models can enable efficient, cost-effective testing of therapeutics, whereas ex vivo systems tend to more accurately predict both positive and negative outcomes observed in vivo.^[10]

Nevertheless, ex vivo models are subject to inherent limitations, including post-mortem alterations in biophysical properties, progressive tissue degradation, limited viability duration, and, in general, the absence or artificial replication of circulation and innervation.^[a] These constraints hinder the models' ability to reproduce long-term or systemic physiological effects.^[5][12] Some of these factors complicate direct comparisons with in vivo systems; for example, measured electric-field values diverge increasingly as the post-mortem interval lengthens.^[12] The boundary between ex vivo and in vitro models remains contested, particularly in the fields of regenerative medicine and tissue engineering, where the terms have been used interchangeably in many studies.^{[13]: 443–444} Modern in vitro models have evolved from simple two-dimensional cultures to sophisticated three-dimensional constructs—such as organoids and organ-on-a-chip devices—that mimic tissue architecture, further blurring the distinction.^[14] Klein and Hutmacher (2024) propose that a model may be classified as ex vivo if it satisfies one or more of the following criteria: it preserves the native structure of the tissue without disrupting its cellular or extracellular components; it maintains near-physiological conditions, such as through artificial perfusion; or it is employed therapeutically in contexts where tissue is removed and subsequently reimplanted. According to these criteria, systems involving extensive reorganization or manipulation—including organoids, organ-on-a-chip, and organotypic cultures—are classified as in vitro, even when they reproduce certain organ-level functions.^{[13]: 448–449}

Techniques

Demonstration of isolation of choroid from the mouse eye^[15]

In situ lung function evaluation, and assessment of total lung capacity (TLC) and basal elastance after performing a recruitment maneuver^[16]

Ex vivo research relies on a range of techniques. Organ perfusion, for instance, involves perfusing isolated organs with oxygenated solutions that mimic blood flow to maintain viability, as seen in ex vivo lung perfusion used to assess donor lungs for transplantation. This method enables researchers to investigate organ-level responses to drugs, disease states, or environmental changes under controlled conditions. By contrast, organ culture traditionally involves maintaining organ sections or small fragments in static or semi-static conditions without active perfusion.^[17][18]

Cell culture involves isolating individual cells from tissues and growing them in a medium enriched with nutrients and growth factors. While these cultures retain some functional characteristics of their tissue of origin, they often exhibit changes in phenotype and gene expression when removed from their native environment. Primary cell cultures, derived directly from tissues, more closely resemble physiological conditions than immortalized cell lines, making them essential for studying cellular behavior, disease mechanisms, and drug effects.^[19][20] Ex vivo microscopy (EVM) uses advanced digital microscopes—such as confocal or optical-coherence devices—to produce microscopic images of fresh tissue, without mounting thin sections on glass slides. Because the tissue stays intact, surgeons can assess tumor margins or examine biopsy samples during surgery.^[21] Computed tomography (CT) is employed in ex vivo research to generate non-destructive, high-resolution imaging of internal structures.^[22] In some cases, ex vivo electroporation, in which an electric field is applied to cells to facilitate the uptake of genetic material, is used to introduce DNA into cells within tissue slices, allowing researchers to study gene expression in a controlled environment.^[23]: 241

Applications

In cardiovascular research, a Langendorff heart preparation involves removing a heart from an organism and perfusing it with a nutrient-rich solution. This method maintains the structural integrity and electrical conduction pathways of the heart, making it suitable for studying phenomena like arrhythmias or drug effects in a more physiologically relevant setting without the complexities of an in vivo model.^[24][25] In dermatological research, human skin organ culture (HSOC) is a technique in which excised human skin is maintained in an artificial medium that preserves its native architecture. HSOC models are employed to study wound healing, drug penetration, and toxicological responses. By retaining the structural complexity of human skin, these models facilitate the investigation of conditions that are not reproducible in animal models,^[6][26] such as keloid formation.^[b] Skin explants from surgical procedures allow researchers to observe early-stage physiological responses to laser treatments in ways that closely resemble in vivo conditions, though processes like re-epithelialization occur more slowly than in living tissue.^[29] In intervertebral disc research, ex vivo models that retain vertebral bone allow for testing potential drugs and investigating loading effects on disc degeneration and repair.^[30]

In translational pharmacology, whole-organ perfusion platforms can re-establish pulsatile blood flow in isolated human organs, enabling direct measurement of absorption, metabolism, and toxicity before first-in-human trials. This system maintains the organs in a viable state, allowing compounds to be tested without the extrapolations required in animal models or cell-based assays, and without the safety concerns of early human exposure. By providing human-specific pharmacokinetic data under near-physiological conditions, these systems support decisions on clinical trial progression and may reduce the need for animal testing.^[31] In biosensing and electroanalytical applications, ex vivo methods offer experimental flexibility unavailable in living systems. While many in vivo experiments favor micro- and nanoelectrodes to minimize invasiveness, larger electrodes are routinely used for specific purposes. Ex vivo approaches, by contrast, permit custom electrode geometries that interface precisely with biological tissues under controlled conditions, without the same constraints on size and invasiveness. This adaptability enables detailed examination of biological analytes and their physiological roles. Ex vivo electroanalytical methods are applied in neuroscience, pharmacology, and biomedical engineering to study neurotransmitter dynamics, metabolic activity, and disease-associated biomarkers.^{[32]: 161–164 [33]: 3–4}

History

Oswald Schmiedeberg

Oscar Langendorff

The foundations of ex vivo experimentation were laid in the 19th century. In 1846, German physiologist Carl Ludwig and his student Carl Wild conducted one of the earliest perfusion studies, connecting the heart of a deceased animal to the common carotid artery of a living donor animal. This configuration allowed the donor's circulation to perfuse the coronary vessels of the excised heart. However, because the heart's viability remained dependent on a living organism rather than an artificial perfusion system, the preparation does not meet the strict criteria for ex vivo experimentation.^[25][34] The earliest known studies involving the perfusion of kidneys outside the native organism were conducted by German physiologist Carl Eduard Loebell, who presented his findings in a doctoral dissertation^[c] in 1849.^[35] In 1866, Russian physiologist Elias von Cyon developed the isolated perfused frog heart preparation at the Carl Ludwig Institute of Physiology in Leipzig, Germany. This method was commonly used during the late 19th century and later served as the basis for the isolated perfused mammalian heart preparation.^[36] In 1876, German physiologist Gustav von Bunge and German pharmacologist Oswald Schmiedeberg demonstrated the synthesis of hippuric acid in the isolated dog kidney.^[35] In 1885, German physiologist Maximilian von Frey and Austrian biologist Max von Gruber, working at the Carl Ludwig Institute of Physiology, constructed an apparatus combining a mechanical pump with an early oxygenator that substituted the function of the heart and lungs in experiments on dogs. This device oxygenated blood outside the body and was a precursor to the heart–lung machine.^[37]

In the 1880s, British physiologist Sydney Ringer developed a salt solution that sustained rhythmic contractions in the isolated frog heart. Later named Ringer's solution, it enabled extended observation of cardiac activity and supported controlled experimental studies on cardiac physiology in isolated preparations.^[38] In 1895, German physiologist Oskar Langendorff introduced a method for isolated heart perfusion involving retrograde flow through the aorta to supply the coronary circulation. The Langendorff preparation allowed for direct measurement of cardiac function and precise control of perfusion parameters while minimizing systemic confounders inherent to in vivo models. It became a widely used technique in the study of cardiac physiology and remains a standard method in cardiovascular research.^[25] At the turn of the 20th century, researchers initiated efforts to preserve animal tissues ex vivo within laboratory settings. Early experiments involved isolating tissues from organisms and transferring them to external media to develop reliable cultivation techniques. These studies aimed not only to maintain cellular viability but also to stimulate tissue growth, often using blood plasma—typically sourced from the same animal—as the medium.^[13]: 444

Portrait of Charles Lindbergh (left) and Alexis Carrel with their perfusion apparatus (1938)

In 1935, French surgeon Alexis Carrel and American aviator Charles Lindbergh unveiled the first closed, sterile perfusion pump. The glass-enclosed, three-chamber device maintained a pulsatile flow of oxygenated perfusate through explanted thyroid glands, keeping them viable for up to three weeks in vitro. Their fragments were then transferred to culture flasks, where they gave rise to actively proliferating cell colonies, verifying ex vivo viability. By equalizing pressure and continuously recirculating the medium, the apparatus proved that long-term organ maintenance outside the body was feasible and laid the groundwork for modern perfusion culture techniques.^{[39][† 1]} In 1953, American surgeon John Heysham Gibbon successfully employed a heart–lung machine during open-heart surgery on a human patient. The procedure demonstrated that an artificial circuit with controlled oxygenation and temperature could temporarily maintain systemic circulation.^[37][40] Throughout the 20th century, ex vivo techniques were adapted for a range of animal models. A notable refinement was the development of the working heart model, in which perfusate enters the left atrium and exits through the aorta, more closely replicating physiological flow conditions. Advances in instrumentation enabled detailed assessments of cardiac function, including pressure–volume relationships, oxygen consumption, and myocardial contractility.^[41][42] The artificial organ field contributed significantly to the advancement of ex vivo systems; for example, the development of hemodialysis relied on a series of ex vivo models designed to support and test extracorporeal circulation technologies.^[13]: 447

Ethical and legal aspects

For broader coverage of this topic, see Medical ethics.

Some ex vivo models may offer ethical benefits by reducing reliance on live-animal experimentation relative to in vivo approaches, enabling researchers to conduct physiologically relevant studies without using whole, living organisms.^[43][44] The Langendorff heart preparation requires the use of live animals, as it involves the excision and immediate perfusion of the heart to preserve viability for experimental analysis. However, adaptations of the technique may reduce the number of animals needed for certain protocols by allowing multiple experimental applications from a single specimen.^[45]

In many jurisdictions worldwide, the use of human-derived tissue in biomedical research is governed by ethical and legal frameworks that require informed consent. In Japan, the Ethical Guidelines for Medical and Biological Research Involving Human Subjects (人を対象とする生命科学・医学系研究に関する倫理指針), implemented in 2021, consolidate previous standards and mandate that researchers obtain informed consent when conducting studies involving human tissues.^{[46][† 2]} In Switzerland, the Federal Act on Research involving Human Beings (Human Research Act, HRA) stipulates that all research involving identifiable human tissue must be approved by an ethics committee. Researchers are required to obtain written informed consent from donors, and documentation concerning the origin of the tissue and the consent procedure must be submitted as part of the ethical review process.^{[47][† 3]}

In the United Kingdom, the legal framework governing the removal, storage, and use of human tissue for research varies by jurisdiction. In England, Wales, and Northern Ireland, the Human Tissue Act 2004 mandates that appropriate consent must be obtained for the removal and use of tissue from both the living and the deceased, unless specific statutory exemptions apply.^{[48][† 4]} The Act includes provisions introduced in response to public health scandals in the 1990s, such as the Alder Hey and Bristol Royal Infirmary cases, in which thousands of children's organs were retained without parental knowledge.^[49] In Scotland, the Human Tissue (Scotland) Act 2006 regulates the removal, retention, and use of human tissue for purposes including transplantation and research. Unlike the 2004 Act, which relies on "appropriate consent", the Scottish legislation is based on the principle of "authorisation" as the legal basis for the use of human tissue.^{[† 5]} The 2006 Act was subsequently amended by the Human Tissue (Authorisation) (Scotland) Act 2019, which introduced a system of deemed authorisation for organ and tissue donation after death.^{[† 6]} In Wales, the Human Transplantation (Wales) Act 2013 further diverged by introducing a system of deemed consent for post-mortem organ and tissue donation.^{[† 7]}

In the United States, federal regulations such as the Common Rule and those enforced by the Food and Drug Administration (FDA) stipulate that researchers must obtain informed consent when conducting studies involving human subjects, including the use of identifiable biological materials. The Health Insurance Portability and Accountability Act (HIPAA) further safeguards the confidentiality of personal health information, including data derived from tissue samples.^{[† 8]}

See also

• List of medical roots, suffixes and prefixes
• Neoclassical compound – Compound words composed from Latin or ancient Greek

Notes

1. Specialized ex vivo models have been developed to preserve native innervation. Although many standard ex vivo intestinal preparations, for example, result in the loss of extrinsic neural connections during tissue removal, certain dissection techniques maintain continuity between the intestinal extrinsic nerves and the spinal cord, thereby preserving physiologically relevant signalling.^[11]
2. In animals, researchers can induce hypertrophic scars, which are somewhat similar to keloids, but keloid formation does not occur. Even in animal models designed to exhibit excessive fibrotic responses through genetic manipulation or specific treatments, the characteristic features of keloids—such as growth beyond the original wound margins and tendency to persist or recur without regression—are not observed.^[27][28]
3. Titled De conditionibus, quibus secretiones in glandulis perficiuntur (transl. 'On the conditions under which secretions are produced in the glands'), the dissertation was presented at the University of Marburg, Germany.^[35] This monograph, written in Latin, is available for browsing on Google Books and the Deutsche Digitale Bibliothek.