Environmental Engineering Reference
In-Depth Information
meaningful endpoints that exactly reflects physiological stress, toxicity, or therapy
or some other phenomenon detectable in vivo. This endpoint detection is very
important to form a link between in vitro and in vivo results. The assurance of cell
assay predictability, biocompatibility with biomaterial parameters needs wisely
judged by quality control standards for cell behavior and biological relevance.
These protocols or procedures are needed to be implemented, compared and
enforced during study.
In vitro cell line studies are used to predict a human response at the administered
dose and to determine relative toxicity in a human or related cell line [
13
]. These
in vitro assays are vital in screening nanoparticles or formulations for cytotoxicity
or adverse biological effects before in vivo testing. These in vitro cell assay
methods could significantly reduce the number of in vivo experiments by giving
flexibility to chose formulation and thereby reduction in use of research animals.
Various in vitro toxicity assays have been utilized to determine the viability and/or
cytotoxicity in cultured cells based on colorimetric measurements or by use of
fluorescent dyes as markers to determine cell viability assessing membrane integ-
rity (i.e. neutral red, calcein AM) or cell metabolism (i.e. MTT, alamar Blue). These
types of assays are commonly used for small molecule, and to limited extent for
nanoparticles. However, there is strong need to use sophisticated techniques like
high-throughput screening techniques to understand in detail the mechanism of
cellular interactions. One of the study done on four carbon-based nanomaterials viz.
CB, SWCNT, C60, and nC60 and one non-carbon-based quantum dots were
evaluated in nanomaterials by nine viability assays and flow cytometry in human
skin epithelial cell line (keratinocytes) supports the concept of high-throughput
screening and multiple assay determination. These cell-based assays must be
validated by imaging techniques like transmission electron microscopy or fluores-
cence microscopy or by chemical marker-based viability assays [
13
]. ESR spin
trapping techniques are employed to identify the formation of ROS.
Many commercial sectors offer thousands of transformed cell lines to choose
from. Many cell lines are deposited within commercial registries (i.e. American
Tissue Type Culture Collection, ATCC) (Fig.
6.3
).
Most commonly, commercial available cell lines are genetically altered or
tissue-harvested (primary cells) either with or without serum. Leukocytes and
immune modulatory cells (e.g. neutrophils, macrophages, and dendritic cells),
phagocytic cells (e.g. monocyte and macrophage phenotypes) have been used as
experimental models in uptake studies. In primary phagocytes, special need to take
care of cell differentiation state (i.e. to terminally differentiated macrophages)
which could significantly alter phagocytic and proliferative tendencies of cell.
Primary cells obtained from tumor biopsy may provide a closer representation of
the tumor cell type. However, 2-D monocultures are generally not representative of
the in vivo case because the culture lacks the spatial interactions and phenotypic/
cell type cross-talk of the actual tumor. Additionally, primary cell culture often
requires animal resources, maintenance fees, and post-harvest animal sacrifice,
raising costs, ethical controversies and significant paperwork for the investigator
(e.g. IACUC and IRB applications).
