Frank Weise

Microbioreactor design for 3-D cell cultivation to create a pharmacological screening system

A biocompatible cell culture environment that enables continued existence of three dimensionally aggregated cells in a polycarbonate‐based scaffold structure was developed. A micro structured polymeric scaffold allows perfusion of cells due to a microporous structure generated by ion track etching and micro thermoforming. Biocompatibility and sterilizability was approved for the whole system. As oxygenation and mass transport within a closed system is most relevant for 3‐D cell culture, two approaches of pumping systems were tested. The human hepatocarcinoma cell line HepG2 was used to examine basic cytological parameters in response to the enviroment. Our data indicate that an actively perfused 3‐D cell culture induces a more differentiated phenotype in HepG2 cells than the 2‐D setup. Thus, our results provide further support to the theory that 3‐D‐cultivated cells display a non‐proliferative behavior. In this respect, 3‐D cultures resemble in vivo conditions more closely. Microreactors are widely applied for organic syntheses, but can also be used for screening applications in drug discovery and medical research. The bioreactor versions presented here were equipped with active fluidic components.

Analysis and comparison of oxygen consumption of HepG2 cells in a monolayer and three‐dimensional high density cell culture by use of a matrigrid®

By the use of a MatriGrid® we have established a three‐dimensional high density cell culture. The MatriGrid® is a culture medium permeable, polymeric scaffold with 187 microcavities. In these cavities (300 μm diameter and 207 μm deep) the cells can growth three‐dimensionally. For these experiments we measured the oxygen consumption of HepG2 cell cultures in order to optimize cultivation conditions. We measured and compared the oxygen consumption, growth rate and vitality under three different cultivation conditions: monolayer, three‐dimensional static and three‐dimensional actively perfused. The results show that the cells in a three‐dimensional cell culture consume less oxygen as in a monolayer cell culture and that the actively perfused three‐dimensional cell culture in the MatriGrid® has a similar growth rate and vitality as the monolayer culture. Biotechnol. Bioeng. 2013; 110:2504–2512.

Multi-photon structuring of native polymers: A case study for structuring natural proteins

The trend of mimicking the real biological world has created an intensive search for methods that are able to engineer 3D‐structured biological environments using nano‐ and micro‐system technologies. Recently published methods show the design of 3D structures by multi‐photon induced polymerization of artificial polymers, such as chemically modified natural polymers. However, limitations of this approach are the long processing time and the fact that no native polymers have been used up to date. In this communication, a case study of multi‐photon structuring of unmodified, native proteins (e.g. collagen and fibrinogen) and liquids, such as natural human blood, or cell culture medium supplements, such as fetal calf serum, is presented. Based on a computer‐assisted process, the structures are polymerized precisely. Even adhesion and gluing of cells with this technique are possible. These encouraging results open new avenues for further inquiry, which are discussed in the paper.

Suspended nanowire web

A complex three-dimensional, nanowire based nanoarchitecture is presented, which can be processed by high-throughput bottom-up procedures without any high-resolution lithography. It combines the benefits of three self-organization mechanisms to produce nanostructures, i.e., the formation of nanoneedles, the droplet formation out of a thin metal film, and the vapor-liquid-solid growth of nanowires. The principle is demonstrated for a silicon based suspended nanowire web. Cell adherence on this assembly was found to be superior to other nanostructures. The possibility of fluid transport beneath the nanowire web enables improved microcatalyst principles and the realization of novel interfaces for biosensing or bioelectronics.

Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance

Current preclinical models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes, including autophagy. Three-dimensional (3D) growth cultures have frequently been proposed to overcome the lack of correlation between two-dimensional (2D) monolayer cell cultures and human tumors in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate shear stress, compound flux and removal of metabolites, e.g., via bioreactor systems, through which culture medium is constantly pumped at a flow rate reflecting physiological conditions. Here we show that both static 3D growth and 3D growth within a bioreactor system modulate key hallmarks of cancer cells, including proliferation and cell death as well as macroautophagy, a recycling pathway often activated by highly proliferative tumors to cope with metabolic stress. The autophagy-related gene expression profiles of 2D-grown cells are substantially different from those of 3D-grown cells and tumor tissue. Autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditions. Three-dimensional cultures depleted of the autophagy mediators BECN1, ATG5 or ATG7 or the transcription factor FOXO3, are more sensitive to cytotoxic treatment. Accordingly, combining cytotoxic treatment with compounds affecting late autophagic flux, such as chloroquine, renders the 3D-grown cells more susceptible to therapy. Altogether, 3D cultures are a valuable tool to study drug response of tumor cells, as these models more closely mimic tumor (patho-)physiology, including the upregulation of tumor relevant pathways, such as autophagy.

In vitro cultivation of biopsy derived primary hepatocytes leads to a more metabolic genotype in perfused 3D scaffolds than static 3D cell culture

The development of reliable systems for testing new compounds for use in the pharmaceutical industry has been a challenging task to date and the concept of a 3D, organotypic cell culture is emerging as a serious alternative to traditional 2D cell cultures or animal testing. We developed a biocompatible 3D cell culture environment that enables the continued existence of cells in a polycarbonate scaffold structure optionally housed in a perfusable bioreactor system. This article focuses on scaffold-based 3D cultivation strategies of biopsy-derived primary human hepatocytes. Cells were examined for whole genome gene expression and for basic cytological parameters. Examining gene networks by Illumina Pathways Analysis revealed that genes associated with metabolic functions are predominantly upregulated under perfusion. Further, we observed individually expressed profiles, which were also reflected by basic cytological parameters such as metabolic activity, albumin production and urea synthesis. With respect to their biotransformation capability, we hypothesize that a perfused 3D culture creates better conditions for the maintenance of primary hepatocytes. For more standardizable experiments with human hepatocytes, we propose the use of a hepatocyte model immortalized by transduction procedures.

Thermoforming techniques for manufacturing porous scaffolds for application in 3D cell cultivation

Within the scientific community, there is an increasing demand to apply advanced cell cultivation substrates with increased physiological functionalities for studying spatially defined cellular interactions. Porous polymeric scaffolds are utilized for mimicking an organ-like structure or engineering complex tissues and have become a key element for three-dimensional (3D) cell cultivation in the meantime. As a consequence, efficient 3D scaffold fabrication methods play an important role in modern biotechnology. Here, we present a novel thermoforming procedure for manufacturing porous 3D scaffolds from permeable materials. We address the issue of precise thermoforming of porous polymer foils by using multilayer polymer thermoforming technology. This technology offers a new method for structuring porous polymer foils that are otherwise available for non-porous polymers only. We successfully manufactured 3D scaffolds from solvent casted and phase separated polylactic acid (PLA) foils and investigated their biocompatibility and basic cellular performance. The HepG2 cell culture in PLA scaffold has shown enhanced albumin secretion rate in comparison to a previously reported polycarbonate based scaffold with similar geometry.

Microfluidic 3D HepG2 cell culture: Reproducing hepatic tumor gene and protein expression in in vitro scaffolds

As for 3D cell culture in general, it is also true for tumor cells that 3D conditions create a spatial microenvironment that is stimulated by concentration gradients of nutrients and gases. Cellular vitality and metabolic activity were examined in 2D and scaffold‐based 3D cultures of HepG2 cells. We compared gene and protein expression levels of tumor‐ and hepato‐specific markers such as alpha‐fetoprotein, albumin, integrin beta 1, insulin‐like growth factor , vascular endothelial growth factor A, and matrix metalloproteinase 11. Although similar biological functions were influenced under 3D static and perfused conditions, their gene expression levels differed. Immunofluorescence stainings detected increased integrin beta 1 expression while alpha‐fetoprotein was downregulated both in static and perfused 3D cultures of HepG2 cells. We hypothesized that improved mass flow in the bioreactors enables the cells to retain their nutrient supply, which is indicated by moderate or even downregulated expression of genes related to angiogenesis and apoptosis. Potential hypoxic stress in static 3D cultures of HepG2 cells resulted in upregulated vascular endothelial growth factor A expression and enhanced caspase 3 activity. Tuning the mass flow in 3D tumor cultures may lead to promising tools to investigate the metabolism and differentiation state of tumor tissues in vitro.