A Brief History of Cell Culture: From Harrison to Organs-on-a-Chip

cells A Brief History of Cell Culture: From Harrison to Organs-on-a-Chip

Abstract: This comprehensive overview of the historical milestones in cell culture underscores key breakthroughs that have shaped the field over time. It begins with Wilhelm Roux's seminal experiments in the eighteen eighties, followed by the pioneering efforts of Ross Granville Harrison, who initiated groundbreaking experiments that fundamentally shaped the landscape of cell culture in the early twentieth century. Carrel's influential contributions, notably the immortalization of chicken heart cells, have marked a significant advancement in cell culture techniques. Subsequently, Johannes Holtfreter, Aron Moscona, and Joseph Leighton introduced methodological innovations in three-dimensional cell culture, initiated by Alexis Carrel, laying the groundwork for future consolidation and expansion of the use of three-dimensional cell culture in different areas of biomedical sciences. The advent of induced pluripotent stem cells by Takahashi and Yamanaka in two thousand six was revolutionary, enabling the reprogramming of differentiated cells into a pluripotent state. Since then, recent innovations have included spheroids, organoids, and organ-on-a-chip technologies, aiming to mimic the structure and function of tissues and organs in vitro, pushing the boundaries of biological modeling and disease understanding. In this review, we overview the history of cell culture shedding light on the main discoveries, pitfalls and hurdles that were overcome during the transition from two-dimensional to three-dimensional cell culture techniques. Finally, we discussed the future directions for cell culture research that may accelerate the development of more effective and personalized treatments.

One. An Overview: The Cell Culture History

Cell culture entails a spectrum of techniques that facilitate the in vitro development of cells, whether of animal or plant origin, thereby isolating them from their native biological context. This approach seeks to partially recapitulate the physicochemical conditions prevailing in the cell's original microenvironment. The art and science of cell culture have enjoyed a rich history, finding application across diverse realms within the biological and biomedical sciences, underscoring their profound methodological significance in dissecting cellular responses to distinct biophysical and biochemical stimuli.

The beginning of cell culture finds its roots in the fields of embryology, biological development, and later, the study of cancer. The earliest documented pursuits can be traced to the late nineteenth and early twentieth centuries. In the eighteen eighties, the renowned physician and microbiologist Robert Koch, then associated with the University of Berlin (Dahlem, Germany), considered the founder of medical bacteriology, was responsible for important contributions regarding the refinement of microorganism cultivation techniques, especially with regard to the identification of the pathogenic agents that cause tuberculosis (Mycobacterium tuberculosis) and cholera (Vibrio cholerae). These discoveries deservedly earned Koch the Nobel Prize in medicine, awarded to him in nineteen zero five. In addition, Dr. Koch proposed a methodological approach widely used to assess the causal relationship between microorganisms and infectious diseases. This approach was called "Koch's Postulates". Some researchers, however, consider the nomenclature "Koch's Postulate" to be strictly incorrect, since Koch would have only improved the postulate previously proposed by his mentor Friedrich Gustav Jakob Henle.

Despite this debate, Koch's most significant contribution to the cell culture knowledge was when he employed gelatin to solidify the culture media. The goal was to facilitate its distribution throughout the culture flask, achieving a consistent and uniform membrane-like layer that covers the whole culture flask surface. This method marked a pivotal enhancement for isolating, identifying, and cultivating individual species of microorganisms in a dish, and represents a cornerstone even nowadays. Furthermore, it is important to highlight that Dr. Koch rigorously emphasized the necessity of sterilizing laboratorial items to minimize or eliminate the possibility of sample contamination, thereby ensuring a high accuracy in experimental results.

Moreover, it is worth mentioning Koch's studies conducted in collaboration with Richard Petri, during their time working together in the Imperial Health Office (Berlin, Germany). Dr. Richard Julius Petri was a German physician and bacteriologist who made significant contributions to microorganism cultivation techniques as Koch's assistant. Alongside Dr. Koch, Dr. Petri established the so-called "Petri's dish", an ubiquitous laboratory apparatus widely used by researchers across the world as a culture flask for microorganisms and/or cell cultivation. Although the nomenclature refers exclusively to Richard Petri, it is important to highlight that the Petri dish was a result of an improvement in the culture dish that Dr. Koch was already using in his research, including those works conducted in partnership with Dr. Petri. However, more than a century later, there is still controversy concerning the nomenclature "Petri's dish" and its true creator.

Still in the eighteen eighties, Wilhelm Roux, a pioneering German experimental embryologist from the University of Halle (Halle, Germany), embarked on groundbreaking experiments involving embryonic cells extracted from avian sources. His work yielded compelling evidence that it was feasible to sustain cellular life beyond the confines of the host organism by immersing them in a saline solution.

Shortly thereafter, the notable legacy of Leo Loeb, a German medical practitioner who later migrated to the United States, came to the fore. His decision to leave Germany was motivated by his dissatisfaction with the nation's nationalistic and militaristic situation. At the Washington University (Seattle, Washington, USA), Loeb emerged as a distinguished experimental pathologist whose groundbreaking contributions in cell culture, transplantation, and hormonal research left an indelible mark on the landscape of medical science. Loeb's profound dedication to research, combined with his visionary approach to humanitarianism, firmly established him as a pivotal figure in the annals of experimental pathology. His enduring influence continues to inspire and guide scientists worldwide, providing the foundational framework for numerous scientific breakthroughs and advancements in the field.

In nineteen zero six, the researcher Ross Granville Harrison (Johns Hopkins University, Baltimore, Maryland, USA) developed pioneering experiments that laid the foundation for cell culture as we know it today. His investigations focused on growing tissue samples in test tubes. Harrison's primary focus was on the study of developing nerve fibers in frogs, where he maintained organ fragments in test tubes containing a liquid medium composed of blood clots, saline solution, and agar. Furthermore, Harrison played a crucial role in the development of the "hanging drop" technique, which involved culturing cells within plasma on the underside of glass slides, creating droplets where the cells gathered. This innovative approach, later validated, continues to be employed in contemporary research, evolving through time with refinements and adaptations.

Harrison's notable work culminated in his publication titled "Observations on the Development of Living Nerve Fibers". In this work, he successfully observed the in vitro development of nerve fibers from a single cell or a cluster over a defined period. However, his research faced a persistent challenge in the form of bacterial contaminations, prompting him to introduce aseptic methodologies. This included the sterilization of surgical materials and the heating of experimental glassware, enabling the conduct of experiments and cell cultivation for extended periods, up to five weeks.

Another luminary in the field of cell culture is Alexis Carrel, a Nobel laureate in medicine in nineteen twelve, acclaimed for his introduction of sutures in surgical procedures. Carrel built upon Harrison's pioneering work from nineteen oh six by developing a method for culturing cells in hanging drops, utilizing glass plate covers. During his investigations, Carrel observed that cells proliferated beyond the confines of the tissue and could be sequentially transferred and manipulated onto new plates. These experiments led to the conception of the "Carrel Flasks," which served as the precursor to contemporary cell culture flasks. Subsequently, while cultivating cardiomyocytes in chicken plasma, Carrel noted that the interaction between cells and the culture medium was directly linked to increased cell proliferation. However, he also discerned that the region closer to the center of the culture exhibited a higher likelihood of necrosis development. To address this challenge, the researcher cultivated tissue fragments on silk threads saturated with plasma, creating a surface where all cells had uniform access to the available nutrients within the culture medium. For the first time, a detailed description of a three-dimensional cell culture was presented.

Also, the fruitful and important partnership between Alexis Carrel and Charles Lindbergh should be highlighted. Lindbergh was responsible for developing methods to separate blood serum from the rest of the blood and for introducing the use of glassware known as "Pyrex Glass" for cell cultivation. The flasks had the crucial advantage that they were resistant to high temperatures, enabling sterilization in autoclaves, and maintaining temperatures between one hundred twenty and one hundred seventy degrees Celsius. Carrel consistently emphasized the need to use sterile materials, an important consideration, since these experiments were conducted before Alexander Fleming discovered the first antibiotic. Around the nineteen thirties, Carrel and Lindbergh published studies describing technologies that supported many experiments until the nineteen eighties, when more sophisticated growth factors, cytokines, and complex culture media were introduced, characterizing the technologies currently used worldwide.

One of Carrel's notable contributions was the isolation and cultivation of one of the first immortalized cell lines derived from chicken embryonic hearts. This was only possible due to the adoption of a strict sterilization methodology and consecutive changes in the culture media involving washing with Riger's solution. This strain underwent hundreds of passages and was maintained until mid-nineteen sixty-four, when it was finalized two years after Carrel's death. The strain described and cultivated by Carrel generated significant interest at the time, and it was established that the cells could survive indefinitely.

The immortalization of cell cultures can be induced by factors such as oncogenic viral infections, radiation, and carcinogenic substances, and has been observed in various cultures throughout the nineteen forties and nineteen sixties. One notable example of immortalized cells is HeLa. These cells, which have become fundamental in scientific research, originated in nineteen fifty-one when Henrietta Lacks was diagnosed with aggressive cervical adenocarcinoma at the Johns Hopkins Hospital in Baltimore. After performing a cervical biopsy, the samples were sent to Dr. George Gay, Director of the Tissue Culture Laboratory. Mary Kubicek, his assistant, noticed that the cells remained viable in a nutrient solution based on chicken plasma and cultured Henrietta Lacks' specimen, resulting in robust, rapidly dividing cell cultures. This remarkable cell line was named HeLa, abbreviated as the initial letters of the patient's name, Henrietta Lacks. It is worth mentioning that more than seventy years later since their isolation, HeLa cells still survive, which is more than twice the lifespan of Henrietta, who passed away in October nineteen fifty-one at the age of thirty-one.

After Carrel's pioneering work, approximately thirty-five years had passed before other researchers began to investigate and improve cell culture techniques. Notable scientists, such as Johannes Holtfreter, Aron Arthur Moscona, and Joseph Leighton, contributed in the advancing and refining of cell culture techniques.

In the field of developmental biology, Johannes Holtfreter, from the University of Heidelberg, described an innovative method that allowed the formation of spherical cell aggregates to prevent cells from adhering to the surface of the culture flasks, thus promoting the tridimensional development of these cells. Later, Holtfreter further refined the techniques previously used by introducing an apparatus that agitated the culture flasks. This facilitated contact between the cells and promoted the diffusion of the surrounding nutrients.

Another notable researcher in the field of developmental biology, Aron Arthur Moscona, made several contributions to refining cell culture techniques. Initially, studies on avian embryonic cells showed that cells from distinct organs did not assemble as a mixed structure. Furthermore, in a subsequent investigation, Moscona designed an experiment where cells derived from the lungs of mice and chicks were cultured into contact, resulting in the formation of cell aggregates after a few days. As a result, Moscona obtained liver and cartilage tissues in vitro. This pioneering work positioned Moscona at the forefront of research on cellular chimeras. In addition, Moscona introduced a technique for cultivating cells using Erlenmeyer flasks under constant agitation. The continuous shaking of the culture flasks was intended to prevent the cells from adhering to the surface while stimulating the formation of cell aggregates in a three-dimensional configuration.

Back in the nineteen fifties, Joseph Leighton, a specialist in histology and cellular pathology, raised a crucial concern about maintaining cellular tissue architecture during development in culture flasks. He noted that despite the remarkable importance of two-dimensional cultures, this technique had significant limitations, especially with regard to the space available for cell development, which was not in line with the natural development of these cells in vivo. In one of his most innovative studies, Leighton cultivated cells and tissue fragments in a three-dimensional matrix made up of a cellulose sponge saturated with plasma obtained from bird embryos. This system was then inserted into a culture flask containing nutrients and subjected to constant agitation. As a result, the study revealed that the three-dimensional arrangement of the cellulose sponge matrix allowed the cells to proliferate and migrate in all directions, more accurately reproducing the behavior of these cells in their organs of origin, in vivo. In addition, these three-dimensional cultures had a significantly larger cell surface area when compared to two-dimensional cell cultures. Based on Leighton's pioneering studies, it became clear that there was a distinction between two-dimensional and three-dimensional cell culture methods, with three-dimensional cell culture systems standing out for their advantages, including greater fidelity in reproducing in vivo cellular development and behavior.

In the early nineteen sixties, Ernst McCulloch (University of Toronto, Toronto, Canada) and James Till (Ontario Cancer Institute, Toronto, Canada) began a series of experiments involving the injection of bone marrow cells into irradiated mice. The authors observed that small nodules formed in the spleens of the mice, directly proportional to the number of bone marrow cells injected. Till and McCulloch termed these nodules "spleen colonies" and postulated that each nodule originated from a single bone marrow cell, perhaps a stem cell. In later work, Till and McCulloch, in collaboration with Andy Becker (undergraduate student) and Lou Siminovitch, from the University of Toronto (Toronto, Canada), published in nineteen sixty-three two articles that represent fundamental milestones for the consolidation of self-renewal capacity and, as a result, the formulation of the concept of bone marrow stem cells.

Another important milestone in the history of cell cultures refers to the work developed, from the nineteen sixties onwards, by the Russian physician Alexander Friedenstein (University of Moscow, Moscow, Russia) which represents cardinal contributions in the discovery and establishment of the concept of mesenchymal stromal/stem cells. From bone marrow cell cultures, Friedenstein and collaborators identified and isolated a subpopulation of non-hematopoietic cells, adherent to culture vials, with a fibroblastoid appearance, with the formation of discrete colonies resulting from clonal multiplication, from a single fibroblastic colony-forming cells, the so-called "Fibroblast Colony Forming Cells" or "colony forming units fibroblastic". In vivo transplantation experiments demonstrated the multipotential nature of Fibroblast Colony Forming Cells, since it was possible to obtain different lineages of mesenchymal/mesodermal origin (osteocytes, chondrocytes, and adipocytes) from a single stromal cell. Cells of mesenchymal origin were later named "marrow stromal stem cells" by Maureen Owen (University of Oxford, Oxford, United Kingdom) and subsequently, as proposed by Arnold Caplan in nineteen ninety-one, the term "mesenchymal stem cells" was adopted. In two thousand five, the International Society for Cellular Therapy proposed that the scientific community adopt, in all written and oral communications, the nomenclature "multipotent mesenchymal stromal cells", but variations in the nomenclature still persist in the literature, such as mesenchymal stem cells, mesenchymal stromal cells, and mesenchymal stromal/stem cells. Finally, the work of Friedenstein and collaborators, especially the partnership established with Maureen Owen, represented pioneering and seminal contributions opening new perspectives in cell therapy and regenerative and translational medicine.

In nineteen sixty-four, Malcolm Steinberg and Stephen A. Roth, both from the University of Princeton (Princeton, New Jersey, United States), proposed the adhesion hypothesis, which posited that the cellular rearrangement was influenced by thermodynamic mediators on different adhesion surfaces. However, this hypothesis gained greater significance only in later years, particularly as cells began to be isolated and studied in greater depth, with a focus on stem cells.

Research involving stem cells accelerated from the nineteen eighties onwards, when various researchers were able to isolate and cultivate pluripotent stem cells derived from mouse embryos. In nineteen eighty-one, Martin Evans and Matthew Kaufman, both from the University of Cambridge (Cambridge, United Kingdom), reported the establishment of cell lines derived from mouse blastocysts, which could differentiate in vitro or, after inoculation into mice, give rise to tumors with cells originating from the three embryonic layers-teratomas. In the same year, in December nineteen eighty-one, Gail R. Martin (University of California, San Francisco, California, United States) published an article in which she described "the establishment of cell lines from normal mouse embryos that form teratocarcinomas when injected into mice". In this work, Martin used the term "embryonic stem cells" for the first time in the literature. It is important to highlight that the establishment of in vitro embryonic stem cell cultures allowed the modification and implantation of these cells in adult females, generating genetically modified mice. Because of these works, Martin John Evans was, together with Mario Capecchi and Oliver Smithies, awarded the Nobel Prize in medicine and physiology in two thousand seven.

About seventeen years after the work of Evans, Kaufman, and Martin, James Thomson's team (University of Wisconsin, Madison, Wisconsin, United States) established, for the first time, the cultivation of human embryonic stem cells obtained from the inner cell mass of blastocysts from human embryos on the fifth day after fertilization. These pluripotent cells, which had high differentiation potential across a broad range of tissues, were characterized by their normal karyotypes and high telomerase activity levels, making them useful for various applications in research and medicine.

A paradigm shift occurred in two thousand six when Kazutoshi Takahashi and Shinya Yamanaka, from the University of Kyoto (Kyoto, Japan), described a method that allowed the reprogramming of already differentiated stem cells, creating the so-called "induced pluripotent stem cells". Researchers were able to obtain induced pluripotent stem cells from adult fibroblasts and mouse embryonic stem cells using only specific markers and growth factors (Sox2, Oct3/4, Klf4, and c-Myc). The result was that induced pluripotent stem cells exhibited properties and characteristics similar to embryonic stem cells, as well as expressing some of the same marker genes.

At the same time, in two thousand seven, James Thomson and his team, from the University of Wisconsin (Madison, Wisconsin, United States), obtained pluripotent cells from differentiated adult human cells (fibroblasts). However, the authors used a different combination of genes (Oct4, Sox2, NANOG, and Lin28) compared to those used by Yamanaka's group. Figure two provides a chronological overview, spotlighting the eminent researchers who have pioneered and refined cell culture methodologies since eighteen eighty-five up to nowadays.

With the capability of generating pluripotent stem cells from adult fibroblasts, there has been a substantial increase in the availability of raw materials for research in cellular biology and development. This advance has spurred remarkable progress and the development of previously unimaginable cell culture techniques. Additionally, as somatic cell reprogramming methodologies have become established, various cell types have been effectively utilized in generating induced pluripotent stem cells, expanding beyond fibroblasts. It is notable that peripheral blood cells and urinary cells offer less invasive procurement methods compared to fibroblasts, which typically necessitate skin biopsies. This advantage in accessibility and non-invasiveness underscores the significance of these alternative sources in induced pluripotent stem cell derivation. Table one showcases the pivotal studies regarding induced pluripotent stem cell derivation from diverse cell sources.