LIFTOSCOPE: development of an automated AI-based module for time-effective and contactless analysis and isolation of cells in microtiter plates.

BACKGROUND: The cultivation, analysis, and isolation of single cells or cell cultures are fundamental to modern biological and medical processes. The novel LIFTOSCOPE technology aims to integrate analysis and isolation into one versatile, fully automated device. METHODS: LIFTOSCOPE's three core technologies are high-speed microscopy for rapid full-surface imaging of cell culture vessels, AI-based semantic segmentation of microscope images for localization and evaluation of cells, and laser-induced forward transfer (LIFT) for contact-free isolation of cells and cell clusters. LIFT transfers cells from a standard microtiter plate (MTP) across an air gap to a receiver plate, from where they can be further cultivated. The LIFT laser is integrated into the optical path of an inverse microscope, allowing to switch quickly between microscopic observation and cell transfer. RESULTS: Tests of the individual process steps prove the feasibility of the concept. A prototype setup shows the compatibility of the microscope stage with the LIFT laser. A specifically designed MTP adapter to hold a receiver plate has been designed and successfully used for material transfers. A suitable AI algorithm has been found for cell selection. CONCLUSION: LIFTOSCOPE speeds up cell cultivation and analysis with a target process time of 10 minutes, which can be achieved if the cell transfer is sped up using a more efficient path-finding algorithm. Some challenges remain, like finding a suitable cell transfer medium. SIGNIFICANCE: The LIFTOSCOPE system can be used to extend existing cell cultivation systems and microscopes for fully automated biotechnological applications.

FEM based simulation of magnetic drug targeting in a multibranched vessel model.

BACKGROUND AND OBJECTIVE: Magnetic drug targeting (MDT) is a promising technology to improve cancer therapy. MDT describes the accumulation of drug loaded superparamagnetic iron oxide nanoparticles (SPIONs) at a desired location, e. g. a tumor, by application of a magnetic field. Here, we evaluate the effectivity of MDT for an endoscopic placement of two different configurations of magnet arrays, i. e. six magnets with same poles facing each other and a Halbach array. Compared to conventional magnet setups outside the body, this endoscopic placement gives the possibility to achieve higher magnetic field gradients inside the tumor. METHODS: For such a MDT concept, we present FEM based simulations of MDT tracing single SPIONs in a 3D geometry of eight multibranched vessels with sizes in the range of capillaries. In these simulations, the effect of the magnetic field gradient as well as of magnet distance to the vessel geometry, magnetic flux density of the magnets, SPIONs hydrodynamic diameter and magnetic moment on the MDT effectivity is calculated. The blood flow is modelled as an incompressible Newtonian fluid and the SPIONs are suspended in the blood flow. Statistical significance of the targeting effectivity results is tested with the Mann-Whitney-U-Test. RESULTS: The results show that the magnetic targeting effectivity is up to 32 % higher than the one calculated without the presence of a magnetic field. In the investigated vessel network, this effect on the targeting effectivity is dependent on the number of local magnetic field maxima that are approached with a high gradient and is noticeable up to 200 µm distance of the magnet to the vessel geometry. CONCLUSIONS: We conclude that for an effective application of MDT, the magnet configuration needs to be placed close to the tumor and should yield a large number of magnetic field maxima that are approached with a high gradient.

Towards automation in biologics production via Raman micro-spectroscopy, laser-induced forward cell transfer and surface-enhanced Raman spectroscopy.

Mammalian cells have become the predominant expression system for the production of biopharmaceuticals due to their capabilities in posttranslational modifications. In recent years, the efficacy of these production processes has increased significantly through technical improvements. However, the state of the art in the development of producer cell lines includes many manual steps and is as such very time and cost consuming. In this study we developed a process combination of Raman micro-spectroscopy, laser-induced forward transfer (LIFT) and surface-enhanced Raman spectroscopy (SERS) as an automated machine system for the identification, separation and characterization of single cell-clones for biopharmaceutical production. Raman spectra showed clear differences between individual antibody-producing and non-producing chinese hamster ovary (CHO) cells after their stable transfection with a plasmid coding for an immunoglobulin G (IgG) antibody. Spectra of producing CHO cells exhibited Raman signals characteristic for human IgG. Individual producing CHO cells were successfully separated and transferred into a multiwell plate via LIFT. Besides, changes in concentration of human IgG in solution were detected via SERS. SERS spectra showed the same peak patterns but differed in their peak intensity. Overall, our results show that identification of individual antibody-producing CHO cells via Raman micro-spectroscopy, cell separation via LIFT and determination of changes in concentrations of overexpressed protein via SERS are suitable and versatile tools for assembling a fully automated system for biopharmaceuticals manufacturing.

Optimized vascular network by stereolithography for tissue engineered skin.

This paper demonstrates the essential and efficient methods to design, and fabricate optimal vascular network for tissue engineering structures based on their physiological conditions. Comprehensive physiological requirements in both micro and macro scales were considered in developing the optimisation design for complex vascular vessels. The optimised design was then manufactured by stereolithography process using materials that are biocompatible, elastic and surface bio-coatable. The materials are self-developed photocurable resin consist of BPA-ethoxylated-diacrylate, lauryl acrylate and isobornylacrylate with Irgacure® 184, the photoinitiator. The optimised vascular vessel offers many advantages: 1) it provides the maximum nutrient supply; 2) it minimises the recirculation areas and 3) it allows the wall shear stress on the vessel in a healthy range. The stereolithography manufactured vascular vessels were then embedded in the hydrogel seeded with cells. The results of in vitro studies show that the optimised vascular network has the lowest cell death rate compared with a pure hydrogel scaffold and a hydrogel scaffold embedded within a single tube in day seven. Consequently, these design and manufacture routes were shown to be viable for exploring and developing a high range complex and specialised artificial vascular networks.

New stereolithographic resin providing functional surfaces for biocompatible three-dimensional printing.

Stereolithography is one of the most promising technologies for the production of tailored implants. Within this study, we show the results of a new resin formulation for three-dimensional printing which is also useful for subsequent surface functionalization. The class of materials is based on monomers containing either thiol or alkene groups. By irradiation of the monomers at a wavelength of 266 nm, we demonstrated an initiator-free stereolithographic process based on thiol-ene click chemistry. Specimens made from this material have successfully been tested for biocompatibility. Using Fourier-transform infrared spectrometry and fluorescent staining, we are able to show that off-stoichiometric amounts of functional groups in the monomers allow us to produce scaffolds with functional surfaces. We established a new protocol to demonstrate the opportunity to functionalize the surface by copper-catalyzed azide-alkyne cycloaddition chemistry. Finally, we demonstrate a three-dimensional bioprinting concept for the production of potentially biocompatible polymers with thiol-functionalized surfaces usable for subsequent functionalization.

Local ultraviolet laser irradiation for gradients on biocompatible polymer surfaces.

Generation of supporting structures, which guide cell growth, is a challenging task in the field of tissue engineering. Cell guidance properties of a scaffold are important in the field of neuronal regeneration. Those guiding structures can provide guidance just by mechanical stimulus or by chemical stimuli like cell signaling molecules. For an enhanced guidance, chemical gradients are under investigation. With this study, we show that ultraviolet laser irradiation is a useful tool to activate polymer surfaces with a high temporal and spatial resolution. We demonstrated that poly(methyl methacrylate) (PMMA) and poly-ε-caprolactone (PCL) can be locally activated and functionalized with amine groups that can be used for immobilization of arginine-glycine-aspartic acid (RGD) peptide. The immobilized RGD was detected by neuronal B35 cells. By defined pulse accumulation functionalization density on the surface can be varied for the generation of gradients. We demonstrated that PMMA and PCL have different process windows for functionalization. Although PMMA has a very small process window for activation, PCL allows the generation of stepwise functionalization. The presented technology can help to develop assays for the analysis of cell migration and neuronal regeneration due to flexible patterning easily realized by changing the irradiation parameters.