A Quantitative, Technology Independent, Fidelity Metric for Evaluating Bioprinted Patterned Co-cultures

The study of cell-cell interactions is crucial in the understanding of cell behaviors such as tumor genesis, proliferation, migration, metastasis, and apoptosis. To break down the complex web of signals in vivo, researchers must replicate some parts of this environment with in vitro tissue test systems, composed of multiple cell types arranged close enough to communicate with their neighbors, i.e. high-resolution co-culture patterns. The field of bioprinting is specifically focused on creating co-culture patterns for the purposes of cell studies, but the sample resolutions of most bioprinting systems are still too coarse to permit cell communication. No way currently exists to compare the sample fidelity between the technologies that have succeeded in creating high-resolution co-culture patterns.. This work introduces a quantitative metric for measuring co-culture patterning fidelity for use in comparing systems or tracking changes in fidelity with experiment conditions. The \u27biopatterning fidelity index\u27 (BFI) measures the performance of a system by fitting a scaled mask of the sample pattern over an image of the printed pattern and classifying the cells as correctly or incorrectly placed. A simple model is also introduced to provide a theoretical upper bound on the expected fidelity. The BFI and model were used to assess the performance of a custom bioprinter system. The performance of the system varied between the different cell types. The results indicate that the post-processing procedures were disturbing the fidelity of the patterns. New procedures should be developed that would not disturb the initial pattern fidelity. The best samples came very close to the model\u27s predicted upper bound. As the number of capable technologies increases, the BFI will provide a quantitative, technology-independent method to assess the fidelity of patterned co-cultures. The last section of this work examines the ability of the bioprinting system to create multiple slides of samples with similar cell distributions. It was shown that cartridges which had been exposed to less usage and cleaning had a more consistent cell output, enabling the bioprinting system to create biological comparable samples

Biofabrication: an overview of the approaches used for printing of living cells

The development of cell printing is vital for establishing biofabrication approaches as clinically relevant tools. Achieving this requires bio-inks which must not only be easily printable, but also allow controllable and reproducible printing of cells. This review outlines the general principles and current progress and compares the advantages and challenges for the most widely used biofabrication techniques for printing cells: extrusion, laser, microvalve, inkjet and tissue fragment printing. It is expected that significant advances in cell printing will result from synergistic combinations of these techniques and lead to optimised resolution, throughput and the overall complexity of printed constructs

Development of a fluidic mixing nozzle for 3D bioprinting

3D bioprinting is a relatively new and very promising field that uses conventional 3D printing techniques and adapts them to print biological materials that are suited for use with cells. These bioprinters can be used to print cells encapsulated within biological ink (bio-ink) to create and customize complex three-dimensional tissues and organs. Our work has focused on developing a new bioprinter nozzle that addresses critical gaps with present-day bioprinters, namely, the lack of standardized, physiologically-relevant biomaterials, and their one nozzle per composition printing capacity. These shortcomings preclude printing a range of cellular and biomaterial compositions (including gradients of cells and matrix components) within a single tissue construct. Type I collagen oligomers, a new soluble collagen subdomain that falls between molecular and fibrillar size scales, are ideally suited for tissue fabrication. This collagen formulation, which is produced according to an ASTM voluntary consensus standard, i) exhibits rapid suprafibrillar self-assembly yielding highly interconnected collagen-fibril matrices resembling those found in the body\u27s tissues, ii) supports cell encapsulation, and iii) allows customized, multi-scale design across the broadest range of tissue architectures and physical properties. These properties, along with its superior physiologic relevance, support the use of this biomaterial in the development of a bioprinting nozzle that is able to address the key gaps in the field of 3D bioprinting. After researching microfluidic mixing devices and current bioprinters, early iterations of a 3D bioprinting nozzle were designed and machined to mix three fundamental reagents required to form a broad array of collagen-fibril matrix compositions, namely oligomeric type I collagen (oligomer), oligomer diluent (diluent), and self-assembly reagent (S.A.R). The nozzle was designed to mix specified proportions of these solutions using a combination of hydrodynamic focusing and twisted channel mixing mechanisms before depositing the selfassembling collagen. Three syringe pumps were used to continuously drive varying flow rates of the three reagents to the nozzle, which allowed for the creation of a broad array of cell and matrix compositions, including fibril-density gradients. To validate nozzle performance, three experiments were conducted to define dispensing volume accuracy and precision, mixing quality, and functional performance of dispensed materials, including cells and matrix. In summary, the integration of standardized self-assembling collagens with this innovative fluidic mixer effectively minimizes the number of printing reservoirs, employs a single dispensing nozzle, and most importantly supports on demand fabrication of various tissue compositions. This advanced 3D bioprinting technology, together with our mechanistic-based tissue engineering design principles, is expected to support customized design and fabrication of complex and scalable tissues for both research and medical applications

Bioprinting with live cells

Tissue engineering is an emerging multidisciplinary field to regenerate damaged or diseased tissues and organs. Traditional tissue engineering strategies involve seeding cells into porous scaffolds to regenerate tissues or organs. However, there are still some challenges such as difficulty in seeding different type of cells spatially into a scaffold, limited oxygen and nutrient delivery and removal of metabolic waste from scaffold and weak cell-adhesion to scaffold material need to be overcome for clinically successful results. Because of those challenges, novel scaffold-free approaches based on cellular self-assembly or three-dimensional (3D) bioprinting have been recently pursued. Bioprinting is a relatively new technology where living cells with or without biomaterials are printed layer-by-layer in order to create 3D living structures. In 3D bioprinting, cell aggregates and hydrogels are termed as bioink used as building blocks that are placed by the bioprinter into precise architecture according to developed computer models. In this chapter, we focus on the scaffold-free, self-assembly based bioprinting approaches and some of the novel developments in this field. This chapter will also discuss the importance as well as the challenges for 3D bioprinting using stem cells. We aim to highlight the importance of the continuous cell printing in order to fabricate 3D biological structures with predefined shapes as being the building blocks of large and complex tissues

Biofabrication: an overview of the approaches used for printing of living cells

The development of cell printing is vital for establishing biofabrication approaches as clinically relevant tools. Achieving this requires bio-inks which must not only be easily printable, but also allow controllable and reproducible printing of cells. This review outlines the general principles and current progress and compares the advantages and challenges for the most widely used biofabrication techniques for printing cells: extrusion, laser, microvalve, inkjet and tissue fragment printing. It is expected that significant advances in cell printing will result from synergistic combinations of these techniques and lead to optimised resolution, throughput and the overall complexity of printed constructs

DEVELOPMENT OF TISSUE ENGINEERED TEST SYSTEMS TO STUDY MAMMARY CELL INTERACTIONS IN VITRO

The work described in this dissertation was conducted in the interdisciplinary research environment of the Clemson University Institute for Biological Interfaces of Engineering. A note at the beginning of each chapter acknowledges, as relevant, collaborating doctoral students and reminds the reader where work from each chapter has been presented or published. The overall goal of this work was to develop tissue engineered test system methodologies to allow the study of mammary cell interactions in vitro. The background, as described in Chapter 1, was published in part in Philosophical Transactions of the Royal Society A in 2010. The studies were designed to encompass both microfabrication technology as well as traditional 3D gel-based macrofabrication techniques, both of which will ultimately be necessary to design and fabricate biologically relevant 3D composite breast tissue cultures. The first step was to assess the effectiveness of microfabrication technology (a custom inkjet bioprinter) to eject cellular and acellular bio-inks into specified two-dimensional patterns on a variety of surfaces. Hence Chapter 2 addresses the overall project feasibility; studies are described wherein printing parameters are modified to identify optimal model conditions. These particular studies were designed to 1) evaluate the effect of stage height on cell viability, 2) identify the relationship between the rate of nozzle firing and the viscosity of a bio-ink, and 3) determine the accuracy of cell placement in a printed co-culture pattern. This work was presented at the 2008 Hilton Head Conference on Regenerative Medicine (stage height) and the 2009 Annual Conference of the Society For Biomaterials (nozzle firing). Chapter 3 addresses one of the major limitations of bioprinting, that of cartridge nozzle clogging, and evaluates the effectiveness of ethylenediaminetetraacetic acid as an anti-scalant and anti-aggregant in 2D high-throughput bioprinting. This work was published in 2009 in the Journal of Tissue Engineering and Regenerative Medicine. Furthermore, Chapters 4 and 5 describe the high resolution capability of the custom bioprinting system, which was demonstrated by 1) printing mono- and co-culture patterns and 2) applying thermal inkjet technology to stain histological samples and cell monolayers, which will be important in the future analysis of test systems. Work described in Chapter 4 was presented in part at the 2009 Annual Meeting and Exposition of the Society For Biomaterials and the 2009 IEEE Engineering in Medicine and Biology Society Conference, while work described in Chapter 5 was presented in part at the 2010 Annual Meeting and Exposition of the Society For Biomaterials. As a final bioprinting-based study, Chapter 6 describes the printing of high-resolution patterns of murine cells in 2D to evaluate paracrine signaling among adipocytes and cancer cells. To achieve this end, D1 and 4T1 cells were printed in co-culture patterns and the effect of 4T1 cells on the proliferation of D1 cells treated with an adipogenic cocktail was evaluated. This work was presented at the 2010 IEEE Engineering in Medicine and Biology Society Conference. Before merging inkjet technology with traditional 3D gel-based culture techniques, 3D gels with incorporated 3D rigid substrates were developed to sustain anchorage dependent stromal cells in a breast tissue co-culture model. As described in Chapter 7, the differences in the activity of stromal cells (adipocytes) seeded on beads versus cells suspended in a gel were determined, as was the effect of adipocytes (seeded on beads and directly in a gel) on mammary epithelial cells. This work will provide a foundation on which tissue test systems with biologically relevant features may be built. Chapter 8 presents work dedicated to education and outreach in tissue engineering. Specifically, a series of classroom teaching modules are presented that can be used to demonstrate basic tissue engineering concepts, such as the effect of the shape of a medical implant on surrounding tissue or the effect of scaffold surface texture on cell attachment. The long-term goal of this work will be to enhance science, technology, engineering, and mathematics education teaching methods and to enhance graduate student communication skills with a non-scientific audience

Cell Printing: A novel method to seed cells onto biological scaffolds

Bioprinting, defined as depositing cells, extracellular matrices and other biologically relevant materials in user-defined patterns to build tissue constructs de novo or to build upon pre-fabricated scaffolds, is among one of the most promising techniques in tissue engineering. Among the various technologies used for Bioprinting, pressure driven systems are most conducive to preserving cell viability. Herein, we explore the abilities of a novel bioprinter - Digilab, Inc.\u27s prototype cell printer. The prototype cell printer (Digilab Inc., Holliston, MA) is an automated liquid handling device capable of delivering cell suspension in user-defined patterns onto standard cell culture substrates or custom-designed scaffolds. In this work, the feasibility of using the cell printer to deliver cell suspensions to biological sutures was explored. Cell therapy using stem cells of various types shows promise to aid healing and regeneration in various ailments, including heart failure. Recent evidence suggests that delivering bone-marrow derived mesenchymal stem cells to the infarcted heart reduces infarct size and improves ventricular performance. Current cell delivery systems, however, have critical limitations such as inefficient cell retention, poor survival, and lack of targeted localization. Our laboratories have developed a method to produce discrete fibrin microthreads that can be bundled to form a suture and attached to a needle. These sutures can then be seeded with bone-marrow derived mesenchymal stem cells to deliver these cells to a precise location within the heart wall, both in terms of depth and surface localization. The efficiency of the process of seeding cells onto fibrin thread bundles (sutures) has previously been shown to be 11.8 ± 3.9 %, suggesting that 88% of the cells in suspension are not used. Considering that the proposed cell-therapy model for treatment of myocardial infarction contemplates use of autologous bone-marrow derived stem cells, an improvement in the efficiency of seeding cells onto the fibrin sutures is highly desirable. The feasibility of using Digilab\u27s prototype cell printer to deliver concentrated cell suspension containing human mesenchymal stem cells (hMSCs) directly onto a fibrin thread bundle was explored in this work, in order to determine if this technology could be adapted to seed cells onto such biological sutures. First the effect of the printing process on the viability of hMSCs was assessed by comparing to cells dispensed manually using a hand-held pipette. The viability of hMSCs 24 hours post-dispensing using the cell printer was found to be 90.9 ± 4.0 % and by manual pipetting was 90.6 ± 8.2 % (p = ns). Thereafter a special bioreactor assembly composed of sterilizable Delrin plastic and stainless steel pins was designed to mount fibrin thread bundles onto the deck of the cell printer, to deliver a suspension containing hMSCs on the bundles. Highly targeted delivery of cell suspension directly onto fibrin thread bundles (average diameter 310 µm) was achieved with the bundle suspended in mid-air horizontally parallel to the printer\u27s deck mounted on the bioreactor assembly. To compare seeding efficiency, fibrin thread bundles were simultaneously seeded with hMSCs using either the cell printer or the current method (tube-rotator method) and incubated for 24 hours. Seeded thread bundles were visualized using confocal microscopy and the number of cells per unit length of the bundle was determined for each group. The average seeding efficiency with the tube rotator method was 7.0 ± 0.03 % while the cell printer was 3.46 ± 2.24% (p = ns). In conclusion, the cell printer was found to handle cells as gently as manual pipetting, preserve their viability, with the added abilities to dispense cells in user-defined patterns in an automated manner. With further development, such as localized temperature, gas and humidity control on the cell printer\u27s deck to aid cell survival, the seeding efficiency is likely to improve. The feasibility of using this automated liquid handling technology to deliver cells to biological scaffolds in specified patterns to develop vehicles for cell therapy was shown in this study. Seeding other cell types on other scaffolds along with selectively loading them with growth factors or multiple cell types can also be considered. In sum, the cell printer shows considerable potential to develop novel vehicles for cell therapy. It empowers researchers with a supervision-free, gentle, patterned cell dispensing technique while preserving cell viability and a sterile environment. Looking forward, de novo biofabrication of tissue replicates on a small scale using the cell printer to dispense cells, extracellular matrices, and growth factors in different combinations is a very realistic possibility

A critical review of current progress in 3D kidney biomanufacturing: advances, challenges, and recommendations

The widening gap between organ availability and need is resulting in a worldwide crisis, particularly concerning kidney transplantation. Regenerative medicine options are becoming increasingly advanced and are taking advantage of progress in novel manufacturing techniques, including 3D bioprinting, to deliver potentially viable alternatives. Cell-integrated and wearable artificial kidneys aim to create convenient and efficient systems of filtration and restore elements of immunoregulatory function. Whilst preliminary clinical trials demonstrated promise, manufacturing and trial design issues and identification of suitable and sustainable cell sources have shown that more development is required for market progression. Tissue engineering and advances in biomanufacturing techniques offer potential solutions for organ shortages; however, due to the complex kidney structure, previous attempts have fallen short. With the recent development and progression of 3D bioprinting, cell positioning and resolution of material deposition in organ manufacture have never seen greater control. Cell sources for constructing kidney building blocks and populating both biologic and artificial scaffolds and matrices have been identified, but in vitro culturing and/or differentiation, in addition to maintaining phenotype and viability during and after lengthy and immature manufacturing processes, presents additional problems. For all techniques, significant process barriers, clinical pathway identification for translation of models to humans, scaffold material availability, and long-term biocompatibility need to be addressed prior to clinical realisation

A critical review of current progress in 3D kidney biomanufacturing: advances, challenges, and recommendations

The widening gap between organ availability and need is resulting in a worldwide crisis, particularly concerning kidney transplantation. Regenerative medicine options are becoming increasingly advanced and are taking advantage of progress in novel manufacturing techniques, including 3D bioprinting, to deliver potentially viable alternatives. Cell-integrated and wearable artificial kidneys aim to create convenient and efficient systems of filtration and restore elements of immunoregulatory function. Whilst preliminary clinical trials demonstrated promise, manufacturing and trial design issues and identification of suitable and sustainable cell sources have shown that more development is required for market progression. Tissue engineering and advances in biomanufacturing techniques offer potential solutions for organ shortages; however, due to the complex kidney structure, previous attempts have fallen short. With the recent development and progression of 3D bioprinting, cell positioning and resolution of material deposition in organ manufacture have never seen greater control. Cell sources for constructing kidney building blocks and populating both biologic and artificial scaffolds and matrices have been identified, but in vitro culturing and/or differentiation, in addition to maintaining phenotype and viability during and after lengthy and immature manufacturing processes, presents additional problems. For all techniques, significant process barriers, clinical pathway identification for translation of models to humans, scaffold material availability, and long-term biocompatibility need to be addressed prior to clinical realisation

3D Bioprinting in Microgravity: Opportunities, Challenges, and Possible Applications in Space

: 3D bioprinting has developed tremendously in the last couple of years and enables the fabrication of simple, as well as complex, tissue models. The international space agencies have recognized the unique opportunities of these technologies for manufacturing cell and tissue models for basic research in space, in particular for investigating the effects of microgravity and cosmic radiation on different types of human tissues. In addition, bioprinting is capable of producing clinically applicable tissue grafts, and its implementation in space therefore can support the autonomous medical treatment options for astronauts in future long term and far-distant space missions. The article discusses opportunities but also challenges of operating different types of bioprinters under space conditions, mainly in microgravity. While some process steps, most of which involving the handling of liquids, are challenging under microgravity, this environment can help overcome problems such as cell sedimentation in low viscous bioinks. Hopefully, this publication will motivate more researchers to engage in the topic, with publicly available bioprinting opportunities becoming available at the International Space Station (ISS) in the imminent future