3 Development of Microstructuring Technologies
3.3 Use of Polycarbonate Foils in Structuration Processes
Establishing Advanced Cell Cultivation Systems 75 canned food or drinking bottles [11,12]. The effects of BPA as an endocrine-disrupting chemical with estrogenic activity (EEDC) have been described as low-dose ranges in nanomolar concentrations inducing biological activity, which have been missed by tra-ditional toxicological testing. BPA is rapidly absorbed into the blood and metabolized primarily in the liver to BPA-glucuronide (BPAG). Besides its weak estrogenic activity, BPA is suspected to be a carcinogen [13]. Kafi et al. observed that BPA at the concen-tration of 300 nM induced oxidative stress and led to cytotoxicity on neuroblastoma cells by modulating a variety of cell signaling pathways. However, current FDA status is that BPA is safe at the very low levels that occur in some foods, but further studies are ongoing [14]. To address the question whether released BPA from PC substrates might negatively affect the outcome of cell culture experiments, there is currently no data available. However, Hiebl et al. compared inserts from polystyrene, polycarbonate and polyetherimid based on ISO10993-5 with the L929 mouse fibroblast cell line [15]. They observed no significant difference in the extracellular LDH activity and cellular energy metabolism. Nevertheless, changes in the cellular morphology were observed between PC, PS and PEI. On PC, they observed homogeneous distribution of vinculin, whereas on PS and PEI it assembled more peripherally. The authors discussed the hydrophobic-ity of the material to influence cell adhesion and thus the assembly of vinculin.
76 Handbook of Polymers for Pharmaceutical Technologies
Having initially demonstrated its power in fabrication of complex microfluidic devices [18–20], hot embossing technologies are now also used to manufacture cell culturing substrates [21,22].
The forming of high aspect ratios demands a high surface quality of the tools and a well controllable temperature throughout the whole process. An additional time- or temperature-related relaxation step after embossing the tool to the material or active mold removing could improve the performance of the method. Ideally, isostatic remov-ing of the material from the tool or peel-off of the material can be facilitated through a gas film injected between the tool and the embossed material.
One of the most critical issues in embossing technology is the durability of the tools.
Silicon and glass tools have a short processing time and a high accuracy. Many users of embossing machines are also familiar with silicon-based standard microstructur-ing techniques and thus produce their tools in-house themselves. Nevertheless, high aspect ratios, wall friction and the resolving shear forces caused by the material flowing in the cavities lead to a high stress of the tools. This is one of the reasons why silicon and glass tools only have a very limited lifetime. Under these circumstances the aspect of a proper connection of the tools to their corresponding baseplate in the machine is essential. Mechanical clamping and gluing are suitable processes to fix the tools.
Mechanical clamping in most cases is used when the tool is a few millimeters thick or reinforced with a thick back plate. This kind of fixture allows fast tool changes and a wide temperature range. Although a good fixture can allow relative movement between the tool and the baseplate caused by the many different heat expansion coefficients of the materials, especially for a tool larger than 50 mm in lateral dimension, this is important. Gluing combines the advantage of a relative movement, the glue acts like an intermediate layer in-between the tool and the baseplate. Also, the lateral backing of the tool with glue helps to prevent ruptures caused by deformation of brittle tool material.
A disadvantage of glued tools is the limited temperature stability of most glues, so the tools in most cases could not be used higher then approx. 200°C with common epoxy glues. The procedure of tool removal is also time consuming, and in most cases the glue must be reduced to ashes.
Medium to mass production processes require metal tools, which could be made of tool steel or any other kinds of metal in the case of common machining. For excellent surface properties of the tools and molds, brass tools are preferable. Due to precision
Figure 3.1 (a) Groove structure for cultivation muscle cells, (b) Pin structure for cultivation of hematopoietic stem cells.
Establishing Advanced Cell Cultivation Systems 77 machining, superior surfaces with optical qualities are achievable. Conventional machining is limited in aspect ratios, structuring depth and resolution of the tools.
One of the most versatile techniques to create metal tools with a high aspect ratio, good surface quality and high resolution is the process combination of silicon-based manu-facturing of a structure positive and the subsequent galvanic refilling of this surface to achieve a metal mold of the piece. This technique is well known under the abbreviation LIGA (lithography and galvanic molding).
3.3.2 Thermoforming
In comparison to organic scaffolds or hydrogels, polymeric scaffolds are accessible to microscopical assessment and to a wide range of processing technologies that provide access to low-cost production. Polymeric foils are also suitable for thermoforming and are an attractive raw material for manufacturing polymeric 3D cell cultivation scaffolds.
For biological application in cell culture, the scaffolds need to have accurate reproduc-ible properties; otherwise experimental results are hardly comparable or even meaning-less. Porous membranes offer advanced functionalities as they allow for a continuous perfusion of the cells through the scaffold’s micropores, leading to enhanced supply of nutrients and oxygen.
The micro-thermoforming technology presented here creates reproducible scaffolds with well-determined proprieties. Due to its single-step manufacturing operation an effective production of scaffolds is possible through parallelization. To ensure high uniformity of the scaffolds, aspects of continuous quality control were implemented.
However, an inherent disadvantage of the common thermoforming process is the dis-ability to mold porous materials, as pressure equalization through the pores occurs dur-ing processdur-ing of the material. This drawback can be overcome by adddur-ing an additional nonporous support foil to transfer the molding pressure to the porous foil under iso-static conditions. In a one-step sandwich process, an effective and parallel production of scaffolds was achieved. A so-called polymer sandwich is molded during the process.
Subsequently, the support foil can be easily removed from the structured porous mem-brane. To establish a proper working process, boundary conditions for the sandwich are the forming temperature of the membrane, the material composition of the sand-wich and the thickness of the two foils. Integrated thermoforming machines (Wickert Presstech, Landau i.d. Pf., Germany), ensure both vacuum and high pressure genera-tion by means of integrated pumps and compressors. All modules are integrated in the compact housing of the machine (Figure 3.2). Local separation of heating and cooling plates enables fast process cycles. Until pressure is applied, the hotplates are separated by insulated springs and guide rails from the cooling block. The latter consists of alu-minum which shows a big thermal capacity in comparison to the hotplate. The thin but rigid hotplate is manufactured from special grade tool steel with a high breaking strain. Before the gas pressure is applied, the press closes the remaining distance of one millimeter between the cooling block and hotplate. A counterforce can be adapted via variation of the applied gas pressure. To align hotplate and cooling block and to regard different thermal strains of the used materials, guide blocks were mounted. This setup significantly reduces particle generation due to shrinkage and friction of the foil during the cooling phase of the process (Figure 3.3).
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Isostatic technology has many advantages like independence from wedge error of the two tool sides, equal force distribution and easy force generation. But an inherent disadvantage of porous materials is the use of a fluid as force transducer within isostatic pressing. The applied fluidic pressure would immediately cross the membrane depend-ing on pore geometry and parameters of the fluid.
In principle there are two techniques to process porous substrates with thermo-forming. A novel method was shown by Giselbrecht et al. [23], who processed pre-ion-beamed foils with thermoforming. Later the formed structures were etched to achieve porous microstructures. The advantage of this method is the easy process setup.
The inherent problem of this process is the annealing of the pre-beamed pore tracks due to the thermal influence during the heating and cooling cycle. (A detailed descrip-tion of the annealing processes is given by Sekhon et al. [24].) This will lead to unequal pore geometry. Current research shows the occurrence of diaphragm-shaped structures centered in the holes. This acts like a bottleneck and increases the fluidic resistance of the whole structure tremendously. Figure 3.4 shows a comparison of annealed and raw porous PC film. The annealing was received after processing the foil according to a time scale which is standard for modified embossing machines.
Figure 3.2 Machine setup of Wickert WLP 1600S in a clean room environment.
Figure 3.3 Basic scheme of the process cycle, (a) insertion of porous PC-foil and transducer foil (FEP), (b) tool closure, heating and evacuation of microstructures and forming gas chamber, (c) forming of structures by high pressure intake, simultaneous closing of forming tool and cooling block, (d) down cooling of the structures, tool opening, removing for forms package and separation of the foils.
Establishing Advanced Cell Cultivation Systems 79
Pores can be developed from the foil by etching under alkaline conditions. In com-parison to the native and untreated foil, the membrane with previous heat treatment shows a centered restriction in the etched pore. Heat treatment is inherent to the ther-moforming process by heating for 10 min in room temperature up to 160°C, then form-ing and coolform-ing for 6 min down to room temperature. This cycle time is caused by the big thermal mass of the hot plates inside a conventional embossing machine. Also, the installed heating power compared to the mass of the hot plate is low. These restrictions increase the fluidic resistance of the foil tremendously. Unfortunately, they are not vis-ible by light microscopic inspection of the transparent foil and could only be inspected with SEM imaging. Figure 3.4C shows an etched foil with a short heat treatment before etching. This example should show the necessity of a fully integrated and adapted pro-cessing of special foils with a thermoforming technology. Starting with a compact and robust machine setup, tool design and geometry, the heat treatment during the process can influence the manufacturing results dramatically.