Quantitative uptake of colloidal particles by cell cultures.

The use of nanotechnologies involving nano- and microparticles has increased tremendously in the recent past. There are various beneficial characteristics that make particles attractive for a wide range of technologies. However, colloidal particles on the other hand can potentially be harmful for humans and environment. Today, complete understanding of the interaction of colloidal particles with biological systems still remains a challenge. Indeed, their uptake, effects, and final cell cycle including their life span fate and degradation in biological systems are not fully understood. This is mainly due to the complexity of multiple parameters which need to be taken in consideration to perform the nanosafety research. Therefore, we will provide an overview of the common denominators and ideas to achieve universal metrics to assess their safety. The review discusses aspects including how biological media could change the physicochemical properties of colloids, how colloids are endocytosed by cells, how to distinguish between internalized versus membrane-attached colloids, possible correlation of cellular uptake of colloids with their physicochemical properties, and how the colloidal stability of colloids may vary upon cell internalization. In conclusion three main statements are given. First, in typically exposure scenarios only part of the colloids associated with cells are internalized while a significant part remain outside cells attached to their membrane. For quantitative uptake studies false positive counts in the form of only adherent but not internalized colloids have to be avoided. pH sensitive fluorophores attached to the colloids, which can discriminate between acidic endosomal/lysosomal and neutral extracellular environment around colloids offer a possible solution. Second, the metrics selected for uptake studies is of utmost importance. Counting the internalized colloids by number or by volume may lead to significantly different results. Third, colloids may change their physicochemical properties along their life cycle, and appropriate characterization is required during the different stages.

Modelling biological cell attachment and growth on adherent surfaces.

A mathematical model, in the form of an integro-partial differential equation, is presented to describe the dynamics of cells being deposited, attaching and growing in the form of a monolayer across an adherent surface. The model takes into account that the cells suspended in the media used for the seeding have a distribution of sizes, and that the attachment of cells restricts further deposition by fragmenting the parts of the domain unoccupied by cells. Once attached the cells are assumed to be able to grow and proliferate over the domain by a process of infilling of the interstitial gaps; it is shown that without cell proliferation there is a slow build up of the monolayer but if the surface is conducive to cell spreading and proliferation then complete coverage of the domain by the monolayer can be achieved more rapidly. Analytical solutions of the model equations are obtained for special cases, and numerical solutions are presented for parameter values derived from experiments of rat mesenchymal stromal cells seeded onto thin layers of collagen-coated polyethylene terephthalate electrospun fibers. The model represents a new approach to describing the deposition, attachment and growth of cells over adherent surfaces, and should prove useful for studying the dynamics of the seeding of biomaterials.

Verification of cell viability in bioengineered tissues and organs before clinical transplantation.

The clinical outcome of transplantations of bioartificial tissues and organs depends on the presence of living cells. There are still no standard operative protocols that are simple, fast and reliable for confirming the presence of viable cells on bioartificial scaffolds prior to transplantation. By using mathematical modeling, we have developed a colorimetric-based system (colorimetric scale bar) to predict the cell viability and density for sufficient surface coverage. First, we refined a method which can provide information about cell viability and numbers in an in vitro setting: i) immunohistological staining by Phalloidin/DAPI and ii) a modified colorimetric cell viability assay. These laboratory-based methods and the developed colorimetric-based system were then validated in rat transplantation studies of unseeded and seeded tracheal grafts. This was done to provide critical information on whether the graft would be suitable for transplantation or if additional cell seeding was necessary. The potential clinical impact of the colorimetric scale bar was confirmed using patient samples. In conclusion, we have developed a robust, fast and reproducible colorimetric tool that can verify and warrant viability and integrity of an engineered tissue/organ prior to transplantation. This should facilitate a successful transplantation outcome and ensure patient safety.