Matthew E. Pepper

Building off-the-shelf tissue-engineered composites

Rapid advances in technology have created the realistic possibility of personalized medicine. In 2000, Time magazine listed tissue engineering as one of the ‘hottest 10 career choices’. However, in the past decade, only a handful of tissue-engineered products were translated to the clinical market and none were financially viable. The reality of complex business planning and the high-investment, high-technology environment was not apparent, and the promise of tissue engineering was overstated. In the meantime, biologists were steadily applying three-dimensional benchtop tissue-culture systems for cellular research, but the systems were gelatinous and thus limited in their ability to facilitate the development of complex tissues. Now, the bioengineering literature has seen an emergence of literature describing biofabrication of tissues and organs. However, if one looks closely, again, the viable products appear distant. ‘Rapid’ prototyping to reproduce the intricate patterns of whole organs using large volumes of cellular components faces daunting challenges. Homogenous forms are being labelled ‘tissues’, but, in fact, do not represent the heterogeneous structure of the native biological system. In 2003, we disclosed the concept of combining rapid prototyping techniques with tissue engineering technologies to facilitate precision development of heterogeneous complex tissue-test systems, i.e. systems to be used for drug discovery and the study of cellular behaviour, biomedical devices and progression of disease. The focus of this paper is on the challenges we have faced since that time, moving this concept towards reality, using the case of breast tissue as an example.

EDTA enhances high-throughput two-dimensional bioprinting by inhibiting salt scaling and cell aggregation at the nozzle surface

Tissue‐engineering strategies may be employed in the development of in vitro breast tissue models for use in testing regimens of drug therapies and vaccines. The physical and chemical interactions that occur among cells and extracellular matrix components can also be elucidated with these models to gain an understanding of the progression of transformed epithelial cells into tumours and the ultimate metastases of tumour cells. The modified inkjet printer may be a useful tool for creating three‐dimensional (3D) in vitro models, because it offers an inexpensive and high‐throughput solution to microfabrication, and because the printer can be easily manipulated to produce varying tissue attributes. We hypothesized, however, that when ink is replaced with a biologically based fluid (i.e. a ‘bio‐ink’), specifically a serum‐free cell culture medium, printer nozzle failure can result from salt scale build‐up as fluid evaporates on the printhead surface. In this study, ethylene diamine tetra‐acetic acid (EDTA) was used as a culture medium additive to prevent salt scaling and cell aggregation during the bioprinting process. The results showed that EDTA, at a concentration typically found in commercially available trypsin solutions (0.53 mM), prevented nozzle failure when a serum‐free culture medium was printed from a nozzle at 1000 drops/s. Furthermore, increasing concentrations of EDTA appeared to mildly decrease aggregation of 4T07 cells. Cell viability studies were performed to demonstrate that addition of EDTA did not result in significant cell death. In conclusion, it is recommended that EDTA be incorporated into bio‐ink solutions containing salts that could lead to nozzle failure. Copyright

Design and implementation of a two-dimensional inkjet bioprinter

Tissue engineering has the potential to improve the current methods for replacing organs and tissues and for investigating cellular process within the scope of a tissue test system. Bioprinting technology can aid in the difficult task of arranging live mammalian cells and biomaterials in viable structures for tissue engineering purposes. This paper describes a system, based on HP26 series print cartridge technology, capable of precisely depositing multiple cell types in precise patterns. The paper discusses the research, design, and implementation of the printing system, which permits control of droplet firing parameters, including firing energy, speed, and spacing. The results demonstrate the system’s fine patterning ability of viable cells, including two-dimensional patterned co-cultures of two cell types. The system has been specifically designed with the flexibility to be extended to print more than two cell types and/or materials simultaneously and to layer printed patterns to form three-dimensional constructs. With these features, the printing system will serve as the foundation for a biofabrication system capable of three-dimensional cell co-cultures, i.e. tissue test systems.

Characterizing the effects of cell settling on bioprinter output.

The time variation in bioprinter output, i.e. the number of cells per printed drop, was studied over the length of a typical printing experiment. This variation impacts the cell population size of bioprinted samples, which should ideally be consistent. The variation in output was specifically studied in the context of cell settling. The bioprinter studied is based on the thermal inkjet HP26A cartridge; however, the results are relevant to other cell delivery systems that draw fluid from a reservoir. A simple mathematical model suggests that the cell concentration in the bottom of the reservoir should increase linearly over time, up to some maximum, and that the cell output should be proportional to this concentration. Two studies were performed in which D1 murine stem cells and similarly sized polystyrene latex beads were printed. The bead output profiles were consistent with the model. The cell output profiles initially followed the increasing trend predicted by the settling model, but after several minutes the cell output peaked and then decreased. The decrease in cell output was found to be associated with the number of use cycles the cartridge had experienced. The differing results for beads and cells suggest that a biological process, such as adhesion, causes the decrease in cell output. Further work will be required to identify the exact process.

Post-bioprinting processing methods to improve cell viability and pattern fidelity in heterogeneous tissue test systems

Bioprinted tissue test systems show promise as a powerful tool for studying cell-cell interaction in heterogeneous, tissue-like co-culture. Several challenges were encountered while attempting to consistently fabricate samples with high viability and pattern fidelity. This paper evaluates four methods for processing samples after bioprinting but prior to adding media for incubation. These methods, composed of various combinations of three techniques meant to promote cell hydration, are evaluated with respect to sample viability and pattern preservation. In the best performing method, Hanks Balanced Salt Solution was applied immediately after fabrication and a collagen overlayer was applied one hour thereafter. The success of this method highlights the ability of the collagen substrate to absorb moisture, which promotes cell health without disturbing the cells printed location. An addendum to the main study is an investigation of the limits of an HP26 print cartridge to deposit cells at a faster rate for the purpose of creating cell layers with densities that approach confluence.

A general purpose driver board for the HP26 ink-jet cartridge with applications to bioprinting

A method for interfacing an HP26 ink-jet cartridge to a computing resource is presented. A general purpose interface will allow new and custom application of ink-jet printing technology. Drive characteristics of the HP26 cartridge are captured and a driver board subsequently developed that permits direct control of nozzle firing timing and properties. Reproduction of the drive signal and satisfactory ink deposition is validated. The application of ink-jet printing technology to bioprinting research is targeted in this work.

A real-time adaptive oxygen transfer rate estimator for metabolism tracking in escherichia coli cultures

Oxygen transfer rate (OTR) is the most significant signal for aerobic bioprocess control, since most microbic metabolic activity relies on oxygen consumption. However, accurate estimation of OTR is challenging due to the difficulty of determining uncertain oxygen transfer parameters and system dynamics. This paper presents an adaptive estimator, which incorporates exhaust gas, stir speed and dissolved oxygen measurements, to predict the real-time OTR. The design of this estimator takes into account the headspace dilution effect, off-gas sensor dynamics and uncertain oxygen transfer parameters. Through simulation the estimated real-time OTR is shown to accurately track quick changes of oxygen demand in the culture. Thus, it can be applied to a variety of controls and estimation purposes, such as determining when the culture is in oxidative or overflow metabolism.

A CMI (cell metabolic indicator)-based controller for achieving high growth rate Escherichia coli cultures.

A large fraction of biopharmaceuticals are produced in Escherichia coli, where each new product and strain currently requires a high degree of growth characterization in benchtop and industrial bioreactors to achieve economical production protocols. The capability to use a standard set of sensors to characterize a system quickly without the need to conduct numerous experiments to determine stable growth rate for the strain would significantly decrease development time. This paper presents a cell metabolic indicator (CMI) which provides better insight into the E. coli metabolism than a growth rate value. The CMI is the ratio of the oxygen uptake rate (OUR) of the culture and the base addition rate (BAR) required to keep pH at a desired setpoint. The OUR and BAR are measured using a off-gas sensor and pH probe, respectively, and thus the CMI can be computed online. Experimental results demonstrate the relationship between CMI and the different cell metabolic states. A previously published model is augmented with acid production dynamics, allowing for comparison of the CMI-based controller with an open-loop controller in simulation. The CMI-based controller required little a priori knowledge about the E. coli strain in order to achieve a high growth rate. Since many different types of cells exhibit similar behaviors, the CMI concept can be extended to mammalian and stem cells.

Thermal inkjet printing for precision histological staining

Abstract Inkjet printing techniques may allow enhanced analysis of tissues. When the tissue available for processing is limited, for example, the amount of information obtained could be increased if different stains could be applied to discrete areas of the same tissue sections. In addition, it may be more desirable to stain an intact tissue rather than sections of tissue. Currently, there are very few methods by which many different stains can be applied in a controlled manner to a single section and/or single block of tissue. This paper presents a liquid deposition system, based on thermal inkjet technology, capable of precisely applying histological processing liquids to form high precision patterns at desired locations on both fixed tissues and cell monolayers. Four experiments were conducted to show the potential of single-drop reagent application, using aniline blue, toluidine blue, fluorescent probes, and an immunofluorescence antibody to create different patterns. Tissue type was shown to affect the overall quality of staining. The flexibility, resolution, and precision of this system have the potential to enable the formulation of new staining techniques to maximize the amount of information gleaned from a given tissue sample.