The use of cell cultures in medical research

Prof. Dr. rer. nat. habil. Peter C. Dartsch, Dipl. Biochemist

  1. Introduction and background

Since its establishment in 2002 close to the university town of Tubingen in south west Germany, Dartsch Scientific GmbH has conducted research and development without animal testing solely using organ-specific cell cultures. Alongside standard procedures the company has also developed numerous test methods of its own which are deployed successfully in a wide range of different fields to investigate active substances or their mixtures. In doing so our aim is not just to examine the agent’s beneficial action but also any possible toxic effects. The possibilities offered by organ-specific cell cultures in medical research will be examined more closely through selected examples. We will also direct our attention to examining products and methods which have so far tended to meet with scepticism from the conventional medical community yet are of considerable interest in certain disciplines such as alternative, complementary and holistic medicine. The individual sections are intentionally presented in chronological order in the form of anecdotes (“from real scientific life”).

  1. What do we mean by cell culture?

Cell culture refers to the cultivation of animal or plant cells outside the organism in a culture medium. In addition to origin (species and organ), a fundamental distinction is made between immortalised cell lines, i.e. cells of a tissue type which can proliferate indefinitely in the course of cultivation and whose characteristics are very stable, and primary cells. The latter are cells, freshly isolated from organs, which have only a limited lifespan in the culture and then become senescent and die or whose characteristics, with increasing in vitro age, differ considerably from the original cell and can no longer be used for further investigations. With most primary cells a critical value is reached around the fourth to eighth passage, i.e. these cells have divided some 8 to 20 times since being isolated. The chief recommendation is to use cell lines whenever possible, for reasons of cost (fig. 1). Cell cultures are now widely used in basic biological and medical research, in the development and manufacture of biotech products and in the production of vaccines and monoclonal antibodies, for example.

  1. Selected examples of the use of organ-specific cell cultures and/or in vitro models from my own practice

3.1. Possible toxicity of amygdalin through release of hydrogen cyanide in an acid environment

In 2006 a legal dispute came before the 11th Senate of the Lower Saxony Administrative Court between the professional association of dispensing chemists of a German federal state and a licensed chemist who had filled capsules with highly purified amygdalin and sold them to cancer patients. It should be explained that amygdalin is used in alternative medicine as a cancer drug or prescribed as an additional remedy following chemotherapy. Amygdalin is a constituent of bitter almonds and apricot stones and, according to current (and still prevailing) thinking, as a cyanogenic glycoside, is said to cleave off volatile and highly toxic hydrogen cyanide during the digestive process. The legal dispute was not concerned with examining the efficacy of amygdalin as a cancer drug but whether the amygdalin sold by the chemist was really of the required purity and whether this amygdalin actually cleaves off hydrogen cyanide during gastrointestinal passage. Animal experiments and tests on human volunteers were out of the question for obvious reasons so finally my company was approached because we had developed a digestive tract model with subsequent cell culture (fig. 2) which was tailor-made for the issues at the heart of this dispute.

After conducting the investigations as commissioned I was then called before the court as an external expert to present the results once again verbally and to answer the judges’ questions. The result of our investigations showed that, even with an empty stomach where the pH of the gastric juice measured 1.8 and a temperature of 37°C, no cyanide is cleaved off the amygdalin so that it is not possible for hydrogen cyanide to be produced either. Alongside chemical analysis of the gastrointestinal juice, a toxicity test was also conducted with fibroblasts and enterocytes. Moreover, the disputed amygdalin was over 97 % pure. The production of hydrogen cyanide from bitter almonds and apricot stones is based on the simultaneous presence of an additional constituent, beta-glucosidase, which fosters the splitting of amygdalin by enzymes in acids.

3.2. Anti-inflammatory effect of New Zealand green-lipped mussel extracts

At roughly the same time a New Zealand company approached Dartsch Scientific and asked if we could develop an in vitro test with cell cultures with which the anti-inflammatory effect of various extracts of green-lipped mussels, which only grow off New Zealand, could be compared with one another. It should be explained that predominantly the gently extracted, highly active mussel oil extracts containing lyprinol, and to a lesser extent the powder, represent one of the most important means of treating chronic joint inflammation conditions such as arthrosis, arthritis, etc.

After several months’ development work taking the scientific literature as our basis, our refined test with functional neutrophils delivered reliable and reproducible results. Human promyelocytes are routinely reproduced in suspension cultures and differentiated to functional neutrophils following treatment over several days with a chemical agent (fig. 3). Just like the neutrophil granulocytes in the blood, these in vitro-differentiated cells are capable of producing free highly reactive superoxide anion radicals in an oxidative burst. The cells not only do this in vivo in the blood to resist microbial pathogens through phagocytosis, they also migrate to inflamed tissue and maintain the inflammatory process by continually producing radicals. If you have a synthetic, plant- or animal-based active substance which disrupts this vicious cycle of constant radical presence, either by inhibiting the production of radicals in the inflamed tissue or by directly neutralising the radicals, then you have a good chance of a potentially anti-inflammatory substance. In the initial phase we validated this test extensively with well-known anti-inflammatory medications such as Ibuprofen, Diclofenac and Meloxicam and other nonsteroidal antirheumatics (NSAR).

It is worth mentioning that preparations made with green-lipped mussels are extremely effective with joint problems in dogs and even horses. This has been repeatedly demonstrated with the latter in our own experience with certain formulations as well as ones we have ourselves tested. This test has so far proved of great service in screening and also in developing pharmaceutical agents as well as animal and human food supplements, cosmetics, etc. and certainly contributed to more than 30 scientific publications. Amongst other things, we have used this procedure to examine a new plant-based toothpaste with effective microorganisms and published the positive results in 2011 in the Deutsche Zahnarztliche Zeitschrift [German Dental Journal]. Although the journal was originally unwilling to accept the article, this very same article was in fact awarded the 2011 prize for most innovative publication of the year by the Deutsche Gesellschaft fur Zahn-, Mund- and Kieferheilkunde [German Society for Dentistry, Oral Hygiene and Orthodontology].

3.3. Cell regeneration and wound healing

Functionality of damaged tissue is restored in vivo through cell regeneration processes. In the case of a previous injury, tissue can be closed and reinforced in what is known as the granulation phase by the predominant cell type in the affected tissue migrating and dividing. This specific phase is simulated in the test system used here. The fibroblasts are seeded in special culture inserts made of silicone with four compartments arranged in a cross. These were previously introduced onto the base of the petri dishes to adhere thereby producing a defined cell-free area. Once a closed cell layer is produced the inserts are carefully removed with forceps. In this way sharply defined cell-free areas are produced between the individual compartments. The cells can now migrate to these cell-free areas and close the cell-free area again through increased cell division (fig. 4). Recolonisation of the cell-free space is continually recorded with time-lapse video microscopy (optional) and the cells are fixed and dyed after 24 hours. Many different test substances have so far been successfully investigated for their cell regeneration properties using this test system.

3.4. Cells of the respiratory tracts at the air-liquid interface

A recent development since 2016 are cytological investigations on a cultivated human lung cell line in submerged culture (active substance is located in the culture medium in which the cells are immersed) or of human primary cells from the upper and lower respiratory tracts. In the case of nasal epithelial cells, these cells can be examined either straight after removal or cultivated on a membrane provided with pores so that the individual isolated cells become more differentiated again. The strategy here is that the basolateral part of the cells projects into the culture medium from where the whole cell is supplied with nutrients. The apical part of the cell, including the cilia newly formed in the course of differentiation, is exposed to the air however. This is precisely the situation we have in our respiratory tracts. A wide range of investigations imitating in-vivo conditions can be conducted in vitro with these test systems in the areas of pharmacology and toxicology. Test substances can be both directed as aerosols over the cells (= local application) and also transported in the culture medium, which assumes the role of the blood here, to the basolateral part of the cells which does not come into contact with respiratory air (= systemic application). In addition to cell vitality, measurement parameters can include ciliary beat frequency using time-lapse microscopy or cell layer morphology in cross-section. These test systems have to date been used successfully to examine new formulations for nose drops and antiviral active ingredients as well as differences in the effect of tobacco cigarettes compared with electronic cigarettes and their liquids on the respiratory tracts.

  1. Current research projects between scientific acceptance and scepticism

What these projects have in common is that, over the past 10 years, I have been confronted as a scientist “hard liner” with products and phenomena which I initially waved away sceptically without closer inspection. But at some point my curiosity really took hold and then it only needed a tiny step to overcome my scepticism and give myself over to new ideas in a completely impartial manner.

4.1. Dowsers and geological interference zones

The first push towards a more receptive way of thinking actually came around 6 years ago with the request from the Verein fur Geobiologie e.V. [Society for Geobiology] to join their scientific advisory body. I did not need to be asked twice and soon I was in the midst of it, hearing about things which made me extremely sceptical. The society has a research centre in Odenwald in which a pronounced geological interference zone has been divined umpteen times by dowsers and surveyed extremely precisely. The reason this interference zone is so pronounced is because faults are present in the rock, it is an intersection point of the Hartmann grid network which covers the whole earth, and also because a powerful subterranean watercourse is present. Sensitive people display marked physiological changes in this place within a very short time. At no little expense an incubator and evaluation station were set up on this precise spot as well as another incubator at an uncontaminated control site just a metre away. Connective tissue cells in appropriate multiple well culture trays were then left for two days in the geological interference zone and the control zone and cell vitality was subsequently determined. The tests were of course conducted in several individual experiments carried out at intervals. The cells reacted to the interference zone with their basal metabolism stimulated by up to 45 %. Differing stimulation areas were also determined in the multiple well culture trays as a result of differing influence of the three interference parameters. Based on the test results no conclusions may be drawn on whether this stimulation at a cellular level leads to reduced wellbeing or even to health impairment. It is proof however that the effect of geological interference zones can be clearly demonstrated at a cellular level even in an entirely neutral scientific test system. The study was published in the March 2014 issue of the journal CO.med.

4.2. Electromagnetic fields and oxidative stress

Most people these days can scarcely imagine life without mobile phones and the associated permanent accessibility. This is particularly true of the younger generation. In a very short time mobile phones have progressed from initially being an exclusive toy to a daily companion found throughout the whole world. Around 7 billion people now use a mobile phone — no other device in human history has experienced such rapid market penetration. Although mobile phones are not the only “culprits” polluting our environment with electromagnetic radiation (besides live power cables, others include DECT phones and WLAN; and even hearing aids), they are the main influence as they are often carried right next to the body or held right up against the ear when in use. The transmitting antenna is fitted right inside the phone and radiation is emitted more or less in a sphere, and so consequently in the direction of the head. We have been examining cellular response to various devices which produce electromagnetic radiation for several years. It has repeatedly emerged that, irrespective of the number of cells killed by radiation, the underlying mechanism is always the same: oxidative stress (fig. 5). As well as direct damage to the cell (cell membrane integrity, DNA, lipids), this also always leads to an increase in programmed cell death (= apoptosis) in the cell population. We are however also examining devices produced by a German manufacturer which are able to compensate specifically for the athermal element of the radiation whereby the effect on the body is significantly reduced.

4.3. Natural spring water and retrospectively informed water and their beneficial effects on health

Water is the basis of all life and over 70 % of the human body also consists of this chemical compound. Until about 2 years ago, to me, water was just plain H2O and wet. It was only good for bathing once it reached a temperature of at least 26°C. Over these past two years I have conducted numerous experiments with water which have nothing to do with imprinted mineral analysis but with the beneficial effects at a cellular level. Despite all my initial scepticism it emerged that not all water is the same and there are considerable differences between the action of conventional still water (actually dead water through reverse osmosis) and living spring water or retrospectively activated or informed tap water. The three terms living, activated and informed used here mean the positive oscillations stored in water which either arise through flowing through the earth’s crust (e.g. with artesian spring water) or are introduced retrospectively into tap water by technical systems. I am not an expert on water; so I cannot give any further explanations regarding these oscillations and how they are stored. However it reminds me very much of the effect of highly diluted homeopathic remedies where no molecules of the active ingredient are still present but probably just specific information in the water which is then transmitted by shaking.

Leaving aside the fact that there is a whole series of products where, in blind studies, I could not detect any difference between the activated water and the initial water, there were a few where I was more than surprised. Not only were these water samples able to stimulate cell vitality and promote regeneration of connective tissue cells, they also had antioxidative and anti-inflammatory potential. Some of these water samples were also able to significantly increase cell metabolism in functional neutrophils and therefore certainly bring about an improvement in microbial pathogen resistance in the blood in vivo. The results obtained in vitro correlate well with clinical investigations of spring and medicinal waters and frequently help explain the sometimes surprising results seen in subjects/patients.

4.4. BICOM® bioresonance device and its effect at a cellular level

In October 2017 Dartsch Scientific GmbH was commissioned to investigate by means of modern methods in cell biology whether there was any evidence of beneficial effects in cultured connective tissue cells as a result of applying BICOM bioresonance. It was only possible to detect beneficial effects following several experimental approaches over a period of a number of months. This is due to the complexity of the method and the sensitivity of the test device as, when used with cell cultures, even the smallest detail in the practical application must be considered and first optimised.

REGUMED loaned a BICOM BICOM optima® mobil device for the investigation. In the final optimisation stage the BICOM Power-Applicator GST71 (BICOM optima®) was also used, housed in a mini incubator at 37°C for the exposure period. The “Pathogens Ai” program series was used as the basis for the study. All three sub-programs were set to 30 minutes’ duration producing a 90 minute exposure cycle. This cycle was repeated twice in succession when exposing the cell cultures such that the cells were exposed for a total of 180 minutes. The sample cup in the top right of the device always contained the same culture medium for all the experiments as well as cell samples which had been pretreated under the same conditions.

We are obviously aware that, when treating humans, the exposure period should not exceed one hour. However, the primary concern in this case was to establish whether the BICOM bioresonance method can produce any cell response which can be considered positive.

In summary using the BICOM BICOM optima® mobil device on cultured connective tissue cells stimulated cell vitality in a pronounced and statistically significant manner (Fig. 6), which can lead to increased physical performance in humans and consequently to improved wellbeing. It also resulted in significantly improved (= shortened) cell regeneration/wound healing by stimulating cell migration and cell division.

  1. Brief resume

Prof. Peter C. Dartsch initially studied chemistry at Darmstadt Technical University and then biochemistry at Tubingen University. In his thesis (1985) and later his doctorate (1989) and post-doctoral lecturing qualification for human physiology (1991) he studied the cultivation of organ-specific primary cells and the development and establishment of cell culture models resembling in vivo conditions at Tubingen University Faculty of Medicine. In 1997 he was appointed adjunct professor. In 2002 he left public sector employment’ leaving his role as deputy managing director of the Institute for Occupational and Social Medicine, and set up Dartsch Scientific GmbH as managing director and sole shareholder. The company develops cell biology test systems, which do not involve animal testing, for use in the field of preclinical research and development. To date, he has published more than 130 scientific papers and book chapters and delivered 150 lectures and poster presentations as well as supervising over 30 medical doctorates. He continues to serve as an external member of Tubingen University Faculty of Medicine.