3D Cell Culture

Protection for the Achilles Heel of Active Substance Research?

  • Fig. 1: This polymer scaffold (A) which was developed at the Ilmenau University of Technology contains 187 highly porous microcavities on an area of 5x5 mm, which are populated with hepatocytes (B, scale in µm) and which can be used in microbioreactors (C) or are suitable as a co-culture insert system (D).Fig. 1: This polymer scaffold (A) which was developed at the Ilmenau University of Technology contains 187 highly porous microcavities on an area of 5x5 mm, which are populated with hepatocytes (B, scale in µm) and which can be used in microbioreactors (C) or are suitable as a co-culture insert system (D).
  • Fig. 1: This polymer scaffold (A) which was developed at the Ilmenau University of Technology contains 187 highly porous microcavities on an area of 5x5 mm, which are populated with hepatocytes (B, scale in µm) and which can be used in microbioreactors (C) or are suitable as a co-culture insert system (D).
  • Tab. 1: Overview of selected 3D cell culture technologies * see Fig. 1

On 19 March 2006, the German newspaper ‘Frankfurter Allgemeine Zeitung' described drug tests for clinical Phase I studies as the "Achilles heel of drug development". In this phase, the active substance is administered to a small group of healthy test candidates in concentrations below the therapeutic dose. The clinical trial is preceded by the preclinical test phase, in the final stage of which the active substance is tested in mammals. After the successful completion of the preclinical phase, approximately 10 % of all active substances fail due to unexpected side effects in humans. These unexpected side effects can have serious consequences for the test participants. What solution strategies exist in order to reduce such "adverse effects"?

What is 3D Cell Culture?
From a historical point of view, 3D cell culture is a relatively recent cultivation model, which is intended to achieve a better quality of information with regard to biological function. The term designates the arrangement of cells in a three-dimensional space under in-vitro conditions. Just as our body and its organs have a spatial orientation; this should also be implemented in cell cultures. The term 3D cell culture includes a wide spectrum of concepts. These can be assigned to the cultivation of cells in hydrogels, in three dimensionally shaped plastic substrates (scaffolds) as cellular aggregates with a freely controllable diameter (spheroids), as well as to tissue sections. All of these approaches promote the creation of a micro-tissue. Actual organs are permeated with capillary blood vessels, which supply even the smallest parts of the particular tissue with nutrients and gases and remove metabolic products.

3D Cell Culture and Its Applicability in the Process of Active Substance Testing
A behaviour similar to that of organs offers advantages if a new drug´s mode of action needs to be tested and conclusions can be drawn with regard to its pharmacokinetic properties as well as pharmacodynamics. The pharmacokinetic behaviour includes characterization of the bioavailability of an active substance as well as its excretion. In addition, for the description of the effectiveness and safety of a preparation (pharmacovigilance) its effect profile as well as its therapeutic bandwidth needs to be known.

For this, precise knowledge of all the parameters prescribed by the regulations are of significant importance for the approval of an active substance. The strength and duration of the effect a substance exerts in the body can be controlled by its form of administration. Should the active substance be absorbed directly via the mucous membranes of the mouth, released by the action of gastric acids or in the gut or better administrated onto the skin?. Various 3D organ models exist for the particular point of entry and effect of the substance, for example the skin, liver, pancreas or the lungs.

The use of 3D cell cultures for pharmacology is a desirable goal, if one considers the health and economic consequences of the recall of a preparation due to liver damage which is induced by active substances (DILI, i.e. drug-induced liver injury). The development costs for an active substance which has gone through the entire development phase including clinical studies, amount on average to a high double-digit million sum. According to details given by the UN, since 1950 more than 600 preparations have been withdrawn due to drug-induced liver injury, or are only approved subject to stringent conditions. The use of innovative cultivation models could also play a role in the determination of toxicity in accordance with the REACH Ordinance of the European Union for compounds used in industry. 3D cell culture attempts to take into account the organ-specific conditions as far as possible and usually utilizes the processing of plastics, which have already proved to be applicable for cell cultures. At present there are a few commercial manufacturers of 3D cell cultivation systems and there are several approaches to its preparation for commercialization. A brief overview of the available models is given in table 1.

Which Regulatory Requirements Should Be Mandatory for 3D Cell Culture?
According to the regulatory rules, pharmacological testing of active substances is carried out in the early preclinical phase on cells which are cultivated in microtitration plates (2D cell cultures). These cell cultures are characterised by the growth of cells on flat surfaces. With their available formats from 6-well to 1536 well plates, which are committed to dimensions of 127.76 mm x 85.48 mm, microtitration plates set high standards with regard to their applicability in automated processes. Handling of these systems is also very simple. In order to be attractive outside of academic research laboratories, 3D cell cultures should be parallizable easy to handle compatible with standard laboratory automation and/or provide a high level of information in comparison with cell cultures which are carried out conventionally in microtitration plates. The main hurdle for the market launch of these novel cultivation systems is their acceptance as an innovation by potential customers, if essential access conditions are not fulfilled. These relate to evidence of the functional and process reliability of the product groups which are to be developed, the validity of the cultivation results which are achieved and compliance with the legally prescribed quality requirements for the components according to ISO 13485.

What Actual Information Have 3D Cell Cultures Provided Up to Now?
To prove the hypotheses of 3D cell culture, not only needs the concept of a new cultivation system to be structurally implemented, but also the biological functionality ought to be ensured. For example, several criteria must be fulfilled for the 3D cultivation of liver cells. These must each be set in relation to conventional 2D cell cultures and the processes in a living organ:

  • How do such microtissues react to active substances, and is the mode of action comparable to the processes in an organ?
  • In comparison with an organ, can the metabolism be improved in comparison 2D cultures?
  • Can 3D cell culture maintain in vivo transporter activities for a drug´s influx and efflux?

In the current scientific literature, there are a variety of publications which confirm that the expectations for the biological criteria are be fulfilled. The recorded data not only relate to simply available cell lines, but have also been recorded for primary human cells. Primary cells, which originate directly from an organ, place high demands on the cultivation conditions. The absorption and excretion of active substances and metabolic products in liver cells essentially depend on the presence of membrane transport proteins. Studies show an increase in the membrane transport activity in the 3D cell culture model. The enzymes of cytochromperoxidases (CYPs) which are important for the liver metabolism are being intensively investigated. These enzymes are of central importance for the metabolism of the active substances of drugs. The effect of 3D cell cultivation on the regulation of these enzymes was demonstrated by various 3D liver cultivation models by several working groups. Last not least, an active substance study with toxic active substance concentrations of the analgesic paracetamol demonstrated an active participation of biomarkers similar to the body. In contrast, these enzymes were not involved in the 2D culture. A trend for the establishment of more complex 3D co-culture models which could provide an improved quality of information is becoming apparent in research. A further important feature to be implemented is the possibility of examining 3D cell cultures under conditions in which there is an active flow of the cultivation medium through the cells in so-called bioreactors, in order to simulate the circulation of blood.

3D cell culture provides promising approaches in order to improve the research of the method of effect of drug candidates. Against the background that essential approval procedures are internationally harmonised, the results of the different 3D cultivation approaches should be intercomparable. From the ranks of the various suppliers, a standard system for 3D culture which best fulfils the requirements of the pharmaceutical industry could become established in the near future.

[1] Materne E.-M. et al.: Lab on a chip 2013
[2] Godoy P. et al.: Arch Toxicol 2013
[3] Fernekorn U. et al.: RSC Advances 2013
[4] Krewski D. et al.: J Toxicol Environ Health B Crit Rev 2010
[5] Morgan S. et al.: Health Policy 2011





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