Evaluation of Biocompatibility Using In Vitro Methods: Interpretation and Limitations - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 143

Current situation Proposed future situation

New materials & surfaces

In vitro tests (ISO10993-5)
(cytotoxicity extract, «contact»)

Level 1: Cell “contact“-Test (ISO10993-5)

Level 2: Extract-Test (modified ISO10993-5)

animal
cell
line

Level 3: Cell contact-Test

In vivo test

Level 4: cell on growth-test
using 3D-Reaggregate

Level 5:Cell differentiation Test

Level 6: Cell-cell competition test

In vivo test (in vivo bioactivity)

C linical trials C linical trials

Fig. 7 Schematic representation of the current and proposed in vitro test battery for assessing
biocompatibility. Schematic drawing of teh bone nicht: modified from 60

extracts. During the five days incubation period, the cell mass of the controls
should increase at least five-fold relative to the initial mass. A deviation of
observed cell mass would indicate that the material compromises the cell pro-
liferation rate. The difference between the three types of extracts enables a
statement of how far an initial toxicity may be of relevance (=[Answer to
questions 2-4).

All samples for the next four levels are incubated in culture medium for 24 h
before cells are seeded on them, ensuring that the protein layer composition is in
equilibrium. Furthermore, at the same time toxic constituents are excluded that are
released in the first 24 h in an initial burst type of release. As the time-point of
measurement, five days after seeding is taken as a reference point but may be
increased depending on the application, target tissue and cells. The subsequent
tests should be feasible for most cell types but for some cell types the proposed test
needs to be adapted.
• Level 3—cell adherence and spreading test: Cells are seeded on top of the test
material at a density such that they reach 50% confluence after five days. Cells
are stained for nuclei, actin and eventually for a typical differentiation marker.
Cell spreading is qualitatively assessed. This test evaluates how far cells are able
to attach to the test material surface, a process which should occur before
apoptosis is induced [ 91 ], and how far the test material supports ''normal'' cell