Working group - a Portrait:
Cultex Laboratories GmbH
The company is concerned with in vitro methods investigating the effects of aerosols on human lung cells. The Cultex Laboratories GmbH was founded by the biologist Prof. Dr. Michaela Aufderheide and the medical scientist and pathologist Prof. Dr. med. Drs. h. c. Ulrich Mohr in 2007. Before that both worked as department leader resp. director at the Institute of Experimental Pathology of the Medical University Hannover and in the following at the Fraunhofer Institute of Toxicology and Experimental Medicine Hannover (formerly Fraunhofer Institute of Toxicology and Aerosol Research).
Research group of the Cultex GmbH.
Image: Cultex Laboratory GmbH
With the foundation, the scientists could realise their dream of an own laboratory in which they can work on their scientific goals. Suitable rooms were found at the Medical Parc Hannover in which they have an area of 600 square meters on hand. Correspondingly the number of staff members has been increased from six to eleven. Beside five scientists inlusive the management, six technical staff members are currently working in the laboratory.
The research ist devided into two scientific categories:
1. development of cell models from cells of the human respiration tract and
2. the investigaton of the effects of airborne substances (aerosols).
For the co-called exposition studies during which the cultivated cells are exposed to the aerosols, the scientists have developed a specialised device, the CULTEX® Radial Flow System (CULTEX® RFS). It is a further development of the CULTEX® glass modules. The new device is a precision instrument which covers the high material requisition regarding to the compatibility of cellular systems and the areophysical conditions. This is nessessary for a reproducible and stabil aerosol exposition.
Experimental construction of the CULTEX Radial Flow System for the exposition with particle-containing atmospheres.
Image: Cultex Laboratories GmbH
Experimental construction of the CULTEX Radial Flow System for the exposition with cigarette smoke.
Image: Cultex Laboratories GmbH
Human bronchial epithelial cells isolated from lung tissue.
Image: Cultex Laboratories GmbH
Investigation process testing the effects of airborne substances
For exposition, cells cultivated on microporous membranes are placed into the module. Here all life securing functions as media supplement and temperature are ensured. On the upper surface, the so-called „air-lifted“ cultures are directly in contact with the surrounding atmosphere. On the lower surface the cells are supplied with nutrients. The sample-carrying module and can now be brought into contact with the top exposition part of the device (hermetically sealed). The test atmosphere (gases, particles or complex mixtures of substances) can be taken up through the module. With regard to the flow distribution (central sampling of the test atmosphere and radial flow distribution) the construction of the upper part of the device as well as special inlet pipes ensure the homogenous distribution of the aerosols on the cells. The exposition is reproducible. Repeats lead to comparable results.
For testing the effects of particle-containing atmospheres, by using the electrostatic precipitation the efficiency of impinge on the cells will get increased significantly which is important for the investigation of nanoparticles.
InVitroJobs asked the managing director and one of the founders of Cultex, Prof. Dr. Michaela Aufderheide, about the current status and the perspectives of their research.
InVitroJobs:
In which scientific approaches can the inhalation exposition system CULTEX RFS be applied?
Prof. Dr. Aufderheide:
In our laboratory the device is primarily used in the field of research and development. The projects which are mainly business sponsored belong to the fundamental research dealing with the development of cell models and exposition studies with cigarette smoke, inhalable therapeutics, gases and particles.
InVitroJobs:
What can be said about the actual status of the Cultex RFS-development? Is the prevalidation process finished? Which results were achieved?
Prof. Dr. Aufderheide:
In priciple the technical concept of the CULTEX® RFS Module is finished. Repeatedly we are confronted with new questions which requires adaptations of the module, e. g. due to specialized test atmospheres or the exposition of complex tissue structures.
Currently a prevalidation study1 of the CULTEX method of direct exposition is carried out by the Federal Ministry of Education and Research (BMBF) (The titel is: in vitro determination of the acute toxicity of inhalative effecting particulate matter after directly exposition of cultivated cells from the human respiration tract). Our partners are the University of Mainz, Institute of Pathology, Repair Lab, and the Institute of Toxicology of the German Armed Forces. The requirements concerning the studies are very high because beside biological questions aerosol physical aspects have to be taken into account for ensuring efficient and workable tests. For this purpose all project partners have been equipped with the same devices (particle press, particle generator etc.), with CULTEX® RFS modules and periphery devices (e.g. flow controller etc.). Some of these devices had to developed for especially this pupose.
Apart from that standard operating procedures (SOP´s)2 were created and within collective training units the workflows were harmonized. From our project partner, seh consulting + services, the experimental results of the particle expositions were analysed statistically and evaluated. The parties involved stand in a permanent dialogue between the executing institutions. The prevalidation study is still running. But we are optimistic that it will be successfull as the results of the various labs are positive.
InVitroJobs:
Which substances can be tested with the model?
Prof. Dr. Aufderheide:
In our prevalidation study we investigate only particle containing atmospheres but – as you can imagine – this is only one of the fields of application.
InVitroJobs:
Which parameters can be tested with the exposition system RFS after the exposition of cell cultures?
Prof. Dr. Aufderheide:
In principle all possible endpoints from the cytotoxicity to the genetic toxicity and up to molecular alterations can be examined. Significant for the collection of different parameters is the focus of the current study.
Naturally the aim of the study has an impact on the choice of the cellular test system. In our lab we only use cell material from the human respiration tract. The range covers cell lines3, primary cell cultures4 and complex built cultures which consists of different populations. For screening studies5 we use mainly cell lines as A549, which can cultivated in a suitable amount and which is characterised by a high stability.
InVitroJobs:
Are there any other scientists who already use the model? Which scientific problem do they want to solve?
Prof. Dr. Aufderheide:
Research groups from Japan and Europe work with the CULTEX® RFS module. They for example use it to analyse the biological effects of emissions from diesel engines and airplane turbines, of gases, cigarette smoke, volatile substances, phosgene as well as inhalable therapeutics. The application spectrum is wide. A extensive infrastructure is nessessary as regard to the technical equipment of an institute because in all cases especially generated atmospheres have to be tested. These are mainly characterised chemically and physically, the potential of the active ingredient effects the selection of the determining parameters after exposition.
InVitroJobs:
Why are you using a liquid covered cell culture when the human lung actually is coated with cilia and mucosal tissue?
Prof. Dr. Aufderheide:
The cell culture inserts are covered in the media only during the first few days after seeding them on the microporous membrane (submerse culture management). During this phase the cells´ growth on the membrane has to be supported. When the cells have built up a dense layer the media is removed from the apical site (from the upper site of the culture). The nutrient delivery is only possible from the basal site (under the membrane). These so-called „air-liquid-interface (ALI) culture management“6 are important for the natural differentiation of the lung epithelial cells into ciliated cells and mucus-producing cells. The supply of the cells with nutrients is ensured via the pores of the membranes. The ALI culture management resembles the conditions of the human lung. This is the basis for simulating realistic conditions of the exposition experiments.
InVitroJobs:
„"A549", "BEAS-2B", "16HBE140-": How can one identify the suitable cell line type which can be used in a test with aerosols?
Prof. Dr. Aufderheide:
The permanent cell lines derive either from cancer cells or they are generated by the genetic manipulation of normal cells. They show some of the characteristics of primary cells, however, they have also properties which are specific for the cell line. Therefore they differ significantly in their suitability for a scientific usage. The extent in which certain cellular aspects can be examined with a single cell line has to be determined in preliminary experiments.
InVitroJobs:
Cell lines or primary cells: Which are suitable „cell systems“ that should be exposed to potentially toxic substances?
Prof. Dr. Aufderheide:
Generally cell lines are more suitable for being used in routine examinations because they enhance the reproducibility of the experiment. Permanent cell lines can be cultivated over a long time without displaying any signs of senescence. Caused by a high division rate as well a high-yield production of cells can be achieved.
Primary cells which are isolated from tissue are best to reflect the in vivo conditions because under suitable culture conditions they can be better differentiated. In the opposite to permanent cell lines primary cells have a restricted lifetime. With an increasing number of passage they lose their ability of differentiation. Additionally the cell yield per passage is very low. Primary cells which derive from different donors often react differently to certain stimuli. Because of the restricted availability and the donor-specific differences, the use of primary cells in routine tests is limited. However, it is recommended to verify the validity of the results of permanent cell lines. Further preliminary tests with primary cells can give information about the suitability of cell lines in specific scientific questions.
InVitroJobs:
Wouldn't it be better to apply a model which simulates the human lung better, for instance as the co-called lung slices7 or the artificial reconstruction of several different cell layers inclusive the use of macrophages8 and dendritic cells9 considerating the defense mechanism against penetrating pathogens as it would occur in nature? Is such an approach intended in your subsequent research?
Prof. Dr. Aufderheide:
The production of cell cultures from lung epithelial cells allows the examination of different processes in this tissue for a longer time frame. For instance it is possible to observe the differentiation of the cells precise and to measure important influence parameters. The differentiation of epithelial cells is an important process during the development of the lung but also after an injury. In the case of a deficient repair process the lung epithelium can be damaged permanently and a chronic disease of the respiration tract can occur. Other in vitro systems as lung slices or tissue spheroids10 for instance constist of already differentiated tissue and are therefore not suitable for such investigations. Furthermore the cells viability often is too restricted and the cultivation is intricate (eventually the additional use of bioreactors is nessessary).
InVitroJobs:
Tissue cuts should be viable for only six month. How long do primary cells resp. co-cultures11 which are used in exposition studies live? Can this affect the results?
Prof. Dr. Aufderheide:
Fully differentiated lung epithelial cells can be cultivated for several month whereby they are well suitable for long-term investigations and repeated expositions. Co-culture systems make it possible to simulate the communication between different cell types. For instance it is known that fibroblasts, which are located in the connective tissue under the epithelial layer of the respiration tract, play an important role during the cellular differentiation and wound healing of the tissue because they produce important regulatory cytokines12 and chemokines13. Co-cultures consisting of lung epithelial cells and fibroblasts can simulate these aspect. Furthermore it is possible to add a third cell type to the co-culture, e. g. defense cells like macrophages. However, it is recommended to work step-by-step in order to be able to precisely investigate and evaluate the biochemical processes.
InVitroJobs:
Is the Cultex exposition system also suitable for triple-cell culture models14?
Prof. Dr. Aufderheide:
Yes.
InVitroJobs:
Beside the objection that in vitro models only give a static impression and need an additional systemic approach for a risk assessment: Can you imagine that your model is suitable to replace the currently conducted animal experiments in the endpoint „repeated dose“, part „inhalation toxicology, in the long run“?
Prof. Dr. Aufderheide:
The political pressure to reduce animal experiments and the progress in the field of replacement methods plead for a future of in vitro methods also as a complement to risk assessment. At the moment animal experiments cannot be waived but the in vitro methods can offer a significant contribution to reduce the number of used, for instance in pre-screening acute toxicity tests. But this depends on the acceptance of these methods by the national and international authorities and if they get approved in the admission procedure. In order to get there the methods have to overcome the obstacles of prevalidation and validation. Hereafter further steps are possible. However, his procedure takes several years and a fast progress is not very likely. But this is the crux of the matter: on one hand in vitro methods are urgently needed on the other hand the way it takes to long and the financial support is unsufficient. On this base it is not possible to establish an efficient strategy to speed up the implementation of such methods.
InVitroJobs:
In the long run will it also be possible to apply the Cultex exposition system to medical issues, e. g. in therapeutical approaches preventing lung diseases?
Prof. Dr. Aufderheide:
Naturally the use of such system is not restricted to the toxicological sector but it can be used for medical purposes. For example the induction of cellular alterations caused by known noxins15 to show special aspects of the disease and point out therapeutic possibilities.
InVitroJobs:
Do you collaborate with medical universities or other facilities? Do you supervise final studies or dissertation works?
Prof. Dr. Aufderheide:
Our basic-oriented research makes it also possible to assign dissertation works which deal with our issues „cell models and exposition“. Here we cooperate with Prof. Scheper and Prof. Kasper from the University of Hannover, Institute of Technical Chemistry from which the works are supervised. But we offer internships and thesis assignments mainly on demand because our field of research is very specialised and not in the focus of the regular universal education.
InVitroJobs:
What do you think about the idea to introduce non-animal methods much more in universal education and to give students the possibility to focus on this scientific area?
Prof. Dr. Aufderheide:
In principle, in vitro methods should be taken more into consideration for teaching in higher education. Non-animal methods have found access to many industrial fields as toxicological studies and they will play an important role in the future.
InVitroJobs:
Thank you for the interview.
Glossary:
1 Prevalidation study: cooperative study under contribution of independent test laboratories targeting the determination whether a method is reliable and reproducible.
2 SOP´s: Standard operation Procedures for stationary, diagnostic or llaboratory procedures.
3 Cell lines: cell lines are cells of one kind of tissue which can reproduce themselves unlimited. With the help of gentechnical methods the cells can be immortalized.
4 Primary cultures: they are prepared immediately after the cell oder tissue sampling. Mostly they are not unlimited reproducible.
5 Screening studies: is a sieve test concentrated on special criteria. A screening study makes it possible to find the most suitable method recognising a disease or ifentifying of a healthy status.
6 Air-liquid-interface (ALI) culture management: the cells are supoplied with nutrients from the lower side of the culture, the liquid table is near the surface of the cell culture so that he interface between the liquid media and the gaseous media is located there.
7 lung slices: Cut mostly from human cancer tissue with a small part of healthy tissue which have to be removed additionally for security reasons. The human tumour material is cut into thin sclices of 200 to 300 micrometers and than bred in cultures.
8 Macrophages: Big phagocytes (cells of the innate immune system).
9 Dendritic cells: Cells of the innate immune system with long extensions sthat looks star-like.
10 Tissue spheroids: three-dimensional tissue globules. The chemical milieu and the cell-cell contacts are different in compare to flat layers.
11 Co-culture system: to study the cell interactions several cell types can be bred together in a culture.
12 Cytokines: were produced from the cells of the immune systems. They navigate the immune response.
13 Chemokines: signal proteins, which attract immune cells to the focus of the inflammation.
14 triple-cell culture model: a cell culture model consisting of three different cell types, for instance A549 epithelial cells, macrophages from precursors of the human blood and dendritic cells. Here, the interaction between a given substance (xenobiotic) and the different cell tyopes can be investigated.
15 Noxins: a substance or a circumstance which have an damaging (diseaese -inducing) effect on the organism or organ of the body.