Search

Cultivating meat from stem cells rather than by raising animals is a promising solution to concerns about the negative externalities of meat production. For cultivated meat to fully mimic conventional meat's organoleptic and nutritional properties, innovations in scaffolding technology are required. Many scaffolding technologies are already developed for use in biomedical tissue engineering. However, cultivated meat production comes with a unique set of constraints related to the scale and cost of production as well as the necessary attributes of the final product, such as texture and food safety. This review discusses the properties of vertebrate skeletal muscle that will need to be replicated in a successful product and the current state of scaffolding innovation within the cultivated meat industry, highlighting promising scaffold materials and techniques that can be applied to cultivated meat development. Recommendations are provided for future research into scaffolds capable of supporting the growth of high‐quality meat while minimizing production costs. Although the development of appropriate scaffolds for cultivated meat is challenging, it is also tractable and provides novel opportunities to customize meat properties., Cultivating meat from cells is a promising solution to the environmental, ethical, health, and food‐security challenges associated with conventional meat production. Cultivated meat will require the development of scalable, low‐cost, and edible or biodegradable scaffolds to support cell growth. This review discusses the unique challenges of cultivated meat scaffolding and highlights promising materials and processing methods worthy of further investigation.

We report an effective strategy to design activatable cell–material interfacing systems enabling photo-modulated cellular entry of cargoes and cell adhesion towards surfaces., Spatio-temporally tailoring cell–material interactions is essential for developing smart delivery systems and intelligent biointerfaces. Here we report new photo-activatable cell–material interfacing systems that trigger cellular uptake of various cargoes and cell adhesion towards surfaces. To achieve this, we designed a novel photo-caged peptide which undergoes a structural transition from an antifouling ligand to a cell-penetrating peptide upon photo-irradiation. When the peptide is conjugated to ligands of interest, we demonstrate the photo-activated cellular uptake of a wide range of cargoes, including small fluorophores, proteins, inorganic (e.g., quantum dots and gold nanostars) and organic nanomaterials (e.g., polymeric particles), and liposomes. Using this system, we can remotely regulate drug administration into cancer cells by functionalizing camptothecin-loaded polymeric nanoparticles with our synthetic peptide ligands. Furthermore, we show light-controlled cell adhesion on a peptide-modified surface and 3D spatiotemporal control over cellular uptake of nanoparticles using two-photon excitation. We anticipate that the innovative approach proposed in this work will help to establish new stimuli-responsive delivery systems and biomaterials.

A common approach to tailoring synthetic hydrogels for regenerative medicine applications involves incorporating RGD cell adhesion peptides, yet assessing the cellular response to engineered microenvironments at the nanoscale remains challenging. To date, no study has demonstrated how RGD concentration in hydrogels affects the presentation of individual cell surface receptors. Here we studied the interaction between human mesenchymal stem cells (hMSCs) and RGD-functionalized poly(ethylene glycol) hydrogels, by correlating macro- and nanoscale single-cell interfacial quantification techniques. We quantified RGD unbinding forces on a synthetic hydrogel using single cell atomic force spectroscopy, revealing that short-term binding of hMSCs was sensitive to RGD concentration. We also performed direct stochastic optical reconstruction microscopy (dSTORM) to quantify the molecular interactions between integrin α5β1 and a biomaterial, unexpectedly revealing that increased integrin clustering at the hydrogel-cell interface correlated with fewer available RGD binding sites. Our complementary, quantitative approach uncovered mechanistic insights into specific stem cell-hydrogel interactions, where dSTORM provides nanoscale sensitivity to RGD-dependent differences in cell surface localization of integrin α5β1. Our findings reveal that it is possible to precisely determine how peptide-functionalized hydrogels interact with cells at the molecular scale, thus providing a basis to fine-tune the spatial presentation of bioactive ligands.

Enzyme-sensitive hydrogels are promising cell delivery vehicles for cartilage tissue engineering. However, a better understanding of their spatiotemporal degradation behavior and its impact on tissue growth is needed. The goal of this study was to combine experimental and computational approaches to provide new insights into spatiotemporal changes in hydrogel crosslink density and extracellular matrix (ECM) growth and how these changes influence the evolving macroscopic properties as a function of time. Hydrogels were designed from aggrecanase-sensitive peptide crosslinks using a simple and robust thiol–norbornene photoclick reaction. To study the influence of variations in cellular activity of different donors, chondrocytes were isolated from either juvenile or adult bovine donors. Initial studies were performed to validate and calibrate the model against experiments. Through this process, two key features were identified. These included spatial variations in the hydrogel crosslink density in the immediate vicinity of the cell and the presence of cell clustering within the construct. When these spatial heterogeneities were incorporated into the computational model along with model inputs of initial hydrogel properties and cellular activity (i.e., enzyme and ECM production rates), the model was able to capture the spatial and temporal evolution of ECM growth that was observed experimentally for both donors. In this study, the juvenile chondrocytes produced an interconnected matrix within the cell clusters leading to overall improved ECM growth, while the adult chondrocytes resulted in poor ECM growth. Overall, the computational model was able to capture the spatiotemporal ECM growth of two different donors and provided new insights into the importance of spatial heterogeneities in facilitating ECM growth. Our long-term goal is to use this model to predict optimal hydrogel designs for a wide range of donors and improve cartilage tissue engineering.

Hydrogels are promising materials for mimicking the extra-cellular environment. Here, we present a simple methodology for the formation of a free-standing viscoelastic hydrogel from the abundant and low cost protein serum albumin. We show that the mechanical properties of the hydrogel exhibit a complicated behaviour as a function of the weight fraction of the protein component. We further use X-ray scattering to shed light on the mechanism of gelation from the formation of a fibrillary network at low weight fractions to interconnected aggregates at higher weight fractions. Given the match between our hydrogel elasticity and that of the myocardium, we investigated its potential for supporting cardiac cells in vitro. Interestingly, these hydrogels support the formation of several layers of myocytes and significantly promote the maintenance of a native-like gene expression profile compared to those cultured on glass. When confronted with a multicellular ventricular cell preparation, the hydrogels can support macroscopically contracting cardiac-like tissues with a distinct cell arrangement, and form mm-long vascular-like structures. We envisage that our simple approach for the formation of an elastic substrate from an abundant protein makes the hydrogel a compelling biomedical material candidate for a wide range of cell types.

Concentrating on the case of poly(ethylene glycol) hydrogels, this paper introduces a methodology that enables a natural integration between the development of a so-called mechanistic model and experimental data relating material's processing to response. In a nutshell, we develop a data-driven modeling component that is able to learn and indirectly infer its own parameters and structure by observing experimental data. Using this method, we investigate the relationship between processing conditions, microstructure and chemistry (cross-link density and polymer-solvent interactions) and response (swelling and elasticity) of non-degradable and degradable PEG hydrogels. We show that the method not only enables the determination of the polymer-solvent interaction parameter, but also it predicts that this parameter, among others, varies with processing conditions and degradation. The proposed methodology therefore offers a new approach that accounts for subtle changes in the hydrogel processing.

Encapsulating chondrocytes in synthetic and degradable hydrogels for cartilage tissue engineering enables tuning of scaffold degradation, but provides no biological cues. Culture medium that recapitulates the physiological osmolarity of the interstitial fluid in cartilage can enhance matrix synthesis in the short term, but long-term benefits remain to be determined. This study investigates the long-term effect of culture medium osmolarity on tissue synthesis using chondrocytes isolated from three skeletally mature bovine donors encapsulated in degradable poly(ethylene glycol) hydrogels. The cell-laden hydrogels were cultured up to 4 weeks in standard chondrocyte-specific medium (330 mOsm) or medium adjusted by sucrose or salts (NaCl and KCl) to reach a physiological osmolarity (400 mOsm). Neocartilaginous matrix synthesis and matrix catabolism were evaluated by quantitative and immunofluorescence methods. Hydrogel degradation kinetics of acellular constructs were not affected by medium osmolarity or osmolyte. Matrix composition was predominantly aggrecan and collagen type II for all conditions. One day after encapsulation, total collagen accumulated in the constructs was increased by 80-90% in 400 mOsm medium, regardless of osmolyte. However, this effect did not persist, and at 4 weeks, total collagen synthesized and released to the medium was more than three times higher in 330 mOsm medium. Medium osmolarity had minimal effects on sulfated glycosaminoglycan content and did not affect catabolic activity. These findings suggest that culture medium at physiological osmolarities may not be beneficial for long-term chondrocyte culture in degradable hydrogels, but that initially culturing chondrocytes at a higher osmolarity may enhance early tissue deposition.

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy., Advances in chemical imaging capability can add to our understanding of complex cellular systems. Here the authors develop a framework for label-free quantitative volumetric Raman spectroscopic imaging and use it to visualize and quantify biomolecules in various 3D cellular systems.

Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well–defined biological ‘blank template’ that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three–dimensional system. We customized the backbone of a Streptococcal collagen–like 2 (Scl2) protein with heparin–binding, integrin–binding, and hyaluronic acid–binding peptide sequences previously shown to modulate chondrogenesis and then cross–linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)– and aggrecanase (ADAMTS4)–cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross–linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross–linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel collagen–mimetic protein, cross–linked via multiple enzymatically degradable peptides, provides a highly adaptable and well defined platform to recapitulate a high degree of biological complexity, which could be applicable to numerous tissue engineering and regenerative medicine applications.

Enzyme-sensitive hydrogels are promising for cell encapsulation and tissue engineering, but result in complex spatiotemporal degradation behavior that is characteristic of reaction-diffusion mechanisms. An experimental and theoretical approach is presented to identify dimensionless quantities that serve as a design tool for engineering enzyme-sensitive hydrogels with controlled degradation patterns by tuning the initial hydrogel properties and enzyme kinetics.

A new cartilage-specific degradable hydrogel based on photoclickable thiol-ene poly(ethylene glycol) (PEG) hydrogels is presented. The hydrogel crosslinks are composed of the peptide, CRDTEGE-ARGSVIDRC, derived from the aggrecanase-cleavable site in aggrecan. This new hydrogel is evaluated for use in cartilage tissue engineering by encapsulating bovine chondrocytes from different cell sources (skeletally immature (juvenile) and mature (adult) donors and adult cells stimulated with proinflammatory lipopolysaccharide (LPS)) and culturing for 12 weeks. Regardless of cell source, a twofold decrease in compressive modulus is observed by 12 weeks, but without significant hydrogel swelling indicating limited bulk degradation. For juvenile cells, a connected matrix rich in aggrecan and collagen II, but minimal collagens I and X is observed. For adult cells, less matrix, but similar quality, is deposited. Aggrecanase activity is elevated, although without accelerating bulk hydrogel degradation. LPS further decreases matrix production, but does not affect aggrecanase activity. In contrast, matrix deposition in the nondegradable hydrogels consists of aggrecan and collagens I, II, and X, indicative of hypertrophic cartilage. Lastly, no inflammatory response in chondrocytes is observed by the aggrecanase-sensitive hydrogels. Overall, it is demonstrated that this new aggrecanase-sensitive hydrogel, which is degradable by chondrocytes and promotes a hyaline-like engineered cartilage, is promising for cartilage regeneration.

Encapsulating chondrocytes in synthetic and degradable hydrogels for cartilage tissue engineering enables tuning of scaffold degradation, but provides no biological cues. Culture medium that recapitulates the physiological osmolarity of the interstitial fluid in cartilage can enhance matrix synthesis in the short term, but long-term benefits remain to be determined. This study investigates the long-term effect of culture medium osmolarity on tissue synthesis using chondrocytes isolated from three skeletally mature bovine donors encapsulated in degradable poly(ethylene glycol) hydrogels. The cell-laden hydrogels were cultured up to 4 weeks in standard chondrocyte-specific medium (330 mOsm) or medium adjusted by sucrose or salts (NaCl and KCl) to reach a physiological osmolarity (400 mOsm). Neocartilaginous matrix synthesis and matrix catabolism were evaluated by quantitative and immunofluorescence methods. Hydrogel degradation kinetics of acellular constructs were not affected by medium osmolarity or osmolyte. Matrix composition was predominantly aggrecan and collagen type II for all conditions. One day after encapsulation, total collagen accumulated in the constructs was increased by 80-90% in 400 mOsm medium, regardless of osmolyte. However, this effect did not persist, and at 4 weeks, total collagen synthesized and released to the medium was more than three times higher in 330 mOsm medium. Medium osmolarity had minimal effects on sulfated glycosaminoglycan content and did not affect catabolic activity. These findings suggest that culture medium at physiological osmolarities may not be beneficial for long-term chondrocyte culture in degradable hydrogels, but that initially culturing chondrocytes at a higher osmolarity may enhance early tissue deposition.

Hydrolytically biodegradable poly(ethylene glycol) (PEG) hydrogels offer a promising platform for chondrocyte encapsulation and tuning degradation for cartilage tissue engineering, but offer no bioactive cues to encapsulated cells. This study tests the hypothesis that a semi-interpenetrating network of entrapped hyaluronic acid (HA), a bioactive molecule that binds cell surface receptors on chondrocytes, and crosslinked degradable PEG improves matrix synthesis by encapsulated chondrocytes. Degradation was achieved by incorporating oligo (lactic acid) segments into the crosslinks. The effects of HA molecular weight (MW) (2.9×10(4) and 2×10(6)Da) and concentration (0.5 and 5mgg(-1)) were investigated. Bovine chondrocytes were encapsulated in semi-interpenetrating networks and cultured for 4weeks. A steady release of HA was observed over the course of the study with 90% released by 4weeks. Incorporation of HA led to significantly higher cell numbers throughout the culture period. After 8days, HA increased collagen content per cell, increased aggrecan-positive cells, while decreasing the deposition of hypertrophic collagen X, but these effects were not sustained long term. Measuring total sulfated glycosaminoglycan (sGAG) and collagen content within the constructs and released to the culture medium after 4weeks revealed that total matrix synthesis was elevated by high concentrations of HA, indicating that HA stimulated matrix production although this matrix was not retained within the hydrogels. Matrix-degrading enzymes were elevated in the low-, but not the high-MW HA. Overall, incorporating high-MW HA into degrading hydrogels increased chondrocyte number and sGAG and collagen production, warranting further investigations to improve retention of newly synthesized matrix molecules.

We investigate holographic optical trapping combined with step-and-repeat maskless projection stereolithography for fine control of 3D position of living cells within a 3D microstructured hydrogel. C2C12 myoblast cells were chosen as a demonstration platform because their development into multinucleated myotubes requires linear arrangements of myoblasts. C2C12 cells are positioned in the monomer solution with multiple optical traps at 1064 nm and then are encapsulated by photopolymerization of monomer via projection of a 512x512 spatial light modulator (SLM) illuminated at 405 nm. High 405 nm sensitivity and complete insensitivity to 1064 nm is enabled by a lithium acylphosphinate (LAP) salt photoinitiator. Use of a polyethylene glycol dimethacrylate (PEGDMA) based monomer is compared to that of polyethylene glycol (PEG) hydrogels formed by thiol-ene photo-click chemistry for patterning structures with cellular resolution, and for maintaining cell viability. Cells patterned in thiol-ene with RGD are shown to retain viability up to 4 days after the trapping and encapsulation procedure. Further, cells patterned in thiol-ene with RGD and a degradable ester link, are shown to fuse, indicating the initial stages of development of multi-nucleated cells.

Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing one's quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth.

Encapsulation of autologous adult cartilage cells (chondrocytes) in hydrolytically degradable hydrogels may provide a clinically viable tissue engineering therapy for replacement of damaged or osteoarthritic cartilage. When designing a tissue engineering scaffold, it is crucial to evaluate adult chondrocytes due to their limited growth potential. The objective for this study was to compare extracellular matrix anabolic and catabolic metabolisms by juvenile and adult chondrocytes in hydrolytically degradable hydrogels. Cells were photo-encapsulated in bimodal degradable hydrogels composed of slow-degrading poly(ethylene glycol) (PEG) and the fast-degrading copolymer oligo(lactic acid)-b-PEG-b-oligo(lactic acid) crosslinks, and cultured through four weeks. Cell density was significantly higher in constructs containing adult cells, contributing to higher glycosaminoglycan content per wet weight. However, juvenile cells exhibited higher collagen content per cell. Immunohistochemical visualization revealed cartilage-specific aggrecan and collagen II deposition by both adult and juvenile cells. Immunohistochemically stained catabolically degraded collagen fragments and western blot-detected degraded aggrecan fragments, especially those associated with an osteoarthritic state, were more abundant in constructs with adult cells. Overall, bimodal degradable hydrogel environments were supportive of viable adult cells. However, major challenges with adult cells include their reduced collagen productivity and high catabolic activity, which may impact the quality of the engineered tissues.

While designing poly(ethylene glycol) hydrogels with high moduli suitable for in situ placement is attractive for cartilage regeneration, the impact of a tighter crosslinked structure on the organization and deposition of the matrix is not fully understood. The objectives of this study were to characterize the composition and spatial organization of new matrix as a function of gel crosslinking and study its impact on chondrocytes in terms of anabolic and catabolic gene expression and catabolic activity. Bovine articular chondrocytes were encapsulated in hydrogels with three crosslinking densities (compressive moduli 60, 320 and 590 kPa) and cultured for 25 days. Glycosaminoglycan production increased with culture time and was greatest in the gels with lowest crosslinking. Collagens II and VI, aggrecan, link protein and decorin were localized to pericellular regions in all gels, but their presence decreased with increasing gel crosslinking. Collagen II and aggrecan expression were initially up-regulated in gels with higher crosslinking, but increased similarly up to day 15. Matrix metalloproteinase (MMP)-1 and MMP-13 expression were elevated (∼25-fold) in gels with higher crosslinking throughout the study, while MMP-3 was unaffected by gel crosslinking. The presence of aggrecan and collagen degradation products confirmed MMP activity. These findings indicate that chondrocytes synthesized the major cartilage components within PEG hydrogels, however, gel structure had a significant impact on the composition and spatial organization of the new tissue and on how chondrocytes responded to their environment, particularly with respect to their catabolic expression.

Objective Significant collagen content and tensile properties are difficult to achieve in tissue-engineered articular cartilage. The aim of this study was to investigate whether treating developing tissue-engineered cartilage constructs with modulators of intracellular Na+ or Ca2+ could increase collagen concentration and construct tensile properties. Methods Inhibitors of Na+ ion transporters and stimulators of intracellular Ca2+ were investigated for their ability to affect articular cartilage development in a scaffoldless, 3-dimensional chondrocyte culture. Using a systematic approach, we applied ouabain (Na+/K+-ATPase inhibitor), bumetanide (Na+/K+/2Cl− tritransporter inhibitor), histamine (cAMP activator), and ionomycin (a Ca2+ ionophore) to tissue-engineered constructs for 1 hour daily on days 10–14 of culture and examined the constructs at 2 weeks or 4 weeks. The gross morphology, biochemical content, and compressive and tensile mechanical properties of the constructs were assayed. Results The results of these experiments showed that 20 μM ouabain, 0.3 μM ionomycin, or their combination increased the tensile modulus by 40–95% compared with untreated controls and resulted in an increased amount of collagen normalized to construct wet weight. In constructs exposed to ouabain, the increased percentage of collagen per construct wet weight was secondary to decreased glycosaminoglycan production on a per-cell basis. Treatment with 20 μM ouabain also increased the ultimate tensile strength of neo-tissue by 56–86% at 4 weeks. Other construct properties, such as construct growth and type I collagen production, were affected differently by Na+ modulation with ouabain versus Ca2+ modulation with ionomycin. Conclusion These data are the first to show that treatments known to alter intracellular ion concentrations are a viable method for increasing the mechanical properties of engineered articular cartilage and identifying potentially important relationships to hydrostatic pressure mechanotransduction. Ouabain and ionomycin may be useful pharmacologic agents for increasing tensile integrity and directing construct maturation.

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The proper biological consequences of the device's oxygen delivery were confirmed in cellular models via a proliferation assay and western analysis of the upregulation of hypoxia inducible transcription factor-1alpha. These experiments serve as a demonstration for the platform as a viable tool to increase experimental throughput and permit novel experimental possibilities in any biomedical research lab.

A microfluidic oxygen delivery device was designed to regulate the exposure of cells in culture to oxygen, facilitating experiments involving oxygen-dependent cell processes. Most of the device is composed of the biocompatible elastomer PDMS (polydimethylsiloxane), including the thin gas-permeable membrane which serves as a diffusive interface between a flowing gas source and the cell culture media. Because cell cultures require a sterile environment, the device must be sterile as well. This study was designed to investigate the effects of common sterilization techniques on the diffusive properties of the device. Devices were sterilized repetitively with autoclaving, UV light, Dri-Clean detergent soaks, or ethanol soaks. Diffusion of oxygen across the device membranes was periodically measured using fluorescent microscopy. After 32 treatments no significant changes were seen in oxygen diffusion over time or in sterilization groups compared to control, meaning that the utility of the device is not adversely affected by repetitive sterilization.

We investigate holographic optical trapping combined with step-and-repeat maskless projection stereolithography for fine control of 3D position of living cells within a 3D microstructured hydrogel. C2C12 myoblast cells were chosen as a demonstration platform since their development into multinucleated myotubes requires linear arrangements of myoblasts. C2C12 cells are positioned in the monomer solution with multiple optical traps at 1064 nm and then encapsulated by photopolymerization of monomer via projection of a 512x512 spatial light modulator illuminated at 405 nm. High 405 nm sensitivity and complete insensitivity to 1064 nm was enabled by a lithium acylphosphinate (LAP) salt photoinitiator. These wavelengths, in addition to brightfield imaging with a white light LED, could be simultaneously focused by a single oil immersion objective. Large lateral dimensions of the patterned gel/cell structure are achieved by x and y step-and-repeat process. Large thickness is achieved through multi-layer stereolithography, allowing fabrication of precisely-arranged 3D live cell scaffolds with micron-scale structure and millimeter dimensions. Cells are shown to retain viability after the trapping and encapsulation procedure.