Nurazhani Abdul Raof

Laser-based direct-write techniques for cell printing

Fabrication of cellular constructs with spatial control of cell location (+/-5 microm) is essential to the advancement of a wide range of applications including tissue engineering, stem cell and cancer research. Precise cell placement, especially of multiple cell types in co- or multi-cultures and in three dimensions, can enable research possibilities otherwise impossible, such as the cell-by-cell assembly of complex cellular constructs. Laser-based direct writing, a printing technique first utilized in electronics applications, has been adapted to transfer living cells and other biological materials (e.g., enzymes, proteins and bioceramics). Many different cell types have been printed using laser-based direct writing, and this technique offers significant improvements when compared to conventional cell patterning techniques. The predominance of work to date has not been in application of the technique, but rather focused on demonstrating the ability of direct writing to pattern living cells, in a spatially precise manner, while maintaining cellular viability. This paper reviews laser-based additive direct-write techniques for cell printing, and the various cell types successfully laser direct-written that have applications in tissue engineering, stem cell and cancer research are highlighted. A particular focus is paid to process dynamics modeling and process-induced cell injury during laser-based cell direct writing.

The Maintenance of Pluripotency Following Laser Direct-Write of Mouse Embryonic Stem Cells

The ability to precisely pattern embryonic stem (ES) cells in vitro into predefined arrays/geometries may allow for the recreation of a stem cell niche for better understanding of how cellular microenvironmental factors govern stem cell maintenance and differentiation. In this study, a new gelatin-based laser direct-write (LDW) technique was utilized to deposit mouse ES cells into defined arrays of spots, while maintaining stem cell pluripotency. Results obtained from these studies showed that ES cells were successfully printed into specific patterns and remained viable. Furthermore, ES cells retained the expression of Oct4 in nuclei after LDW, indicating that the laser energy did not affect their maintenance of an undifferentiated state. The differentiation potential of mouse ES cells after LDW was confirmed by their ability to form embryoid bodies (EBs) and to spontaneously become cell lineages representing all three germ layers, revealed by the expression of marker proteins of nestin (ectoderm), Myf-5 (mesoderm) and PDX-1 (endoderm), after 7 days of cultivation. Gelatin-based LDW provides a new avenue for stem cell patterning, with precision and control of the cellular microenvironment.

Bioengineering embryonic stem cell microenvironments for exploring inhibitory effects on metastatic breast cancer cells.

The recreation of an in vitro microenvironment to understand and manipulate the proliferation and migration of invasive breast cancer cells may allow one to put a halt to their metastasis capacity. Invasive cancer cells have been linked to embryonic stem (ES) cells as they possess certain similar characteristics and gene signatures. Embryonic microenvironments have the potential to reprogram cancer cells into a less invasive phenotype and help elucidate tumorigenesis and metastasis. In this study, we explored the feasibility of reconstructing embryonic microenvironments using mouse ES cells cultured in alginate hydrogel and investigated the interactions of ES cells and highly invasive breast cancer cells in 2D, 2&1/2D, and 3D cultures. Results showed that mouse ES cells inhibited the growth and tumor spheroid formation of breast cancer cells. The mouse ES cell microenvironment was further constructed and optimized in 3D alginate hydrogel microbeads, and co-cultured with breast cancer cells. Migration analysis displayed a significant reduction in the average velocity and trajectory of breast cancer cell locomotion compared to control, suggesting that bioengineered mouse ES cell microenvironments inhibited the proliferation and migration of breast cancer cells. This study may act as a platform to open up new options to understand and harness tumor cell plasticity and develop therapeutics for metastatic breast cancer.

Laser direct-write of single microbeads into spatially-ordered patterns

Fabrication of heterogeneous microbead patterns on a bead-by-bead basis promotes new opportunities for sensors, lab-on-a-chip technology and cell-culturing systems within the context of customizable constructs. Laser direct-write (LDW) was utilized to target and deposit solid polystyrene and stem cell-laden alginate hydrogel beads into computer-programmed patterns. We successfully demonstrated single-bead printing resolution and fabricated spatially-ordered patterns of microbeads. The probability of successful microbead transfer from the ribbon surface increased from 0 to 80% with decreasing diameter of 600 to 45 µm, respectively. Direct-written microbeads retained spatial pattern registry, even after 10 min of ultrasonication treatment. SEM imaging confirmed immobilization of microbeads. Viability of cells encapsulated in transferred hydrogel microbeads achieved 37 ± 11% immediately after the transfer process, whereas randomly-patterned pipetted control beads achieved a viability of 51 ± 25%. Individual placement of >10 µm diameter microbeads onto planar surfaces has previously been unattainable. We have demonstrated LDW as a valuable tool for the patterning of single, micrometer-diameter beads into spatially-ordered patterns.

Bioengineering Embryonic Stem Cell Microenvironments for the Study of Breast Cancer

Breast cancer is the most prevalent disease amongst women worldwide and metastasis is the main cause of death due to breast cancer. Metastatic breast cancer cells and embryonic stem (ES) cells display similar characteristics. However, unlike metastatic breast cancer cells, ES cells are nonmalignant. Furthermore, embryonic microenvironments have the potential to convert metastatic breast cancer cells into a less invasive phenotype. The creation of in vitro embryonic microenvironments will enable better understanding of ES cell-breast cancer cell interactions, help elucidate tumorigenesis, and lead to the restriction of breast cancer metastasis. In this article, we will present the characteristics of breast cancer cells and ES cells as well as their microenvironments, importance of embryonic microenvironments in inhibiting tumorigenesis, convergence of tumorigenic and embryonic signaling pathways, and state of the art in bioengineering embryonic microenvironments for breast cancer research. Additionally, the potential application of bioengineered embryonic microenvironments for the prevention and treatment of invasive breast cancer will be discussed.

Convergent mechanisms in pluripotent stem cells and cancer: Implications for stem cell engineering

Stem cells and cancer cells share certain characteristics, including the capacity to self‐renew, differentiatie, and undergo epithelial‐to‐mesenchymal transition (EMT). The mechanisms underlying tumorigenesis retain similarities with processes in normal stem cell development. Comprehensive analysis and comparison of cancer cell and stem cell development will advance the study of cancer progression, enabling development of effective strategies for cancer treatment. In this review article, we first examine the convergence of outcome, cellular communication, and signaling pathways active in pluripotent stem cells and cancer cells. Next, we detail how stem cell engineering is able to mimic in vivo microenvironments. These efforts can help better identify stem cell‐cancer cell interactions, elucidated dysregulated pluripotent signaling pathways occurring in cancer, revealed new factors that restrict tumorigenesis and metastasis potential, and reprogrammed cancer cells to a less aggressive phenotype. The potential of stem cell engineering to enhance cancer research is tremendous and may lead to alternative therapeutic options for aggressive cancers.

Precise Patterning of Mouse Embryonic Stem Cells on Glass Cover Slips for High-Throughput Analysis Using Laser Direct-Write

Engineering a microenvironment where the growth substrate and distance between cells are controlled is highly desirable to understand how cellular interactions affect stem cell differentiation. Laser direct-write (LDW) allows rapid and precise placement of living cells via computer-aided design/computer-aided manufacturing (CAD/CAM) control. Application of this technique to study the effects of various stem cell microenvironments on differentiation requires a high-throughput experimental setup [1]. Recently, our lab has developed a gelatin-based LDW method for the precise patterning of sensitive cell types, such as mouse embryonic stem cells (mESCs), at a resolution of about 5 μm [2]. Although viable mESCs were successfully printed with maintained pluripotency, this technique required cells to be patterned onto polystyrene Petri dishes [2,3], which may limit high-throughput efficiency. Moreover, the use of polystyrene Petri dishes requires large quantities of culture medium and is not convenient for biological analysis of mESC differentiation. Therefore, the objective of this study was to adapt the LDW method, without altering its prior success, to transfer patterns of viable mESCs to glass cover slips. However, this adaptation to cover slips could not be achieved through simple downscaling due to the unique challenges of providing sufficient moisture for viable cell transfer while maintaining pattern registry on a cover slip. Once cells have been laser patterned, cover slips can then be moved to a 24-well plate so that separate sets of laser patterned cells can be analyzed in parallel for higher experimental throughput utilizing fewer resources to maintain the cells.Copyright