Chemically defined production of engineered cardiac tissue microspheres from hydrogel-encapsulated pluripotent stem cells.

Chemically defined, suspension culture conditions are a key requirement in realizing clinical translation of engineered cardiac tissues (ECTs). Building on our previous work producing functional ECT microspheres through differentiation of biomaterial encapsulated human induced pluripotent stem cells (hiPSCs), here we establish the ability to use chemically defined culture conditions, including stem cell media (E8) and cardiac differentiation media (chemically defined differentiation media with three components, CDM3). A custom microfluidic cell encapsulation system was used to encapsulate hiPSCs at a range of initial cell concentrations and diameters in the hybrid biomaterial, poly(ethylene glycol)-fibrinogen (PF), for the formation of highly spherical and uniform ECT microspheres for subsequent cardiac differentiation. Initial microsphere diameter could be tightly controlled, and microspheres could be produced with an initial diameter between 400 and 800 µm. Three days after encapsulation, cardiac differentiation was initiated through small molecule modulation of Wnt signaling in CDM3. Cardiac differentiation occurred resulting in in situ ECT formation; results showed that this differentiation protocol could be used to achieve cardiomyocyte (CM) contents greater than 90%, although there was relatively high variability in CM content and yield between differentiation batches. Spontaneous contraction of ECT microspheres initiated between Days 7 and 10 of differentiation and ECT microspheres responded to electrical pacing up to 1.5 Hz. Resulting CMs had well-defined sarcomeres and the gap junction protein, connexin 43, and had appropriate temporal changes in gene expression. In summary, this study demonstrated the proof-of-concept to produce functional ECT microspheres with chemically defined media in suspension culture in combination with biomaterial support of microsphere encapsulated hiPSCs.

Establishment of a tissue-engineered colon cancer model for comparative analysis of cancer cell lines.

To overcome the limitations of in vitro two-dimensional (2D) cancer models in mimicking the complexities of the native tumor milieu, three-dimensional (3D) engineered cancer models using biomimetic materials have been introduced to more closely recapitulate the key attributes of the tumor microenvironment. Specifically, for colorectal cancer (CRC), a few studies have developed 3D engineered tumor models to investigate cell-cell interactions or efficacy of anti-cancer drugs. However, recapitulation of CRC cell line phenotypic differences within a 3D engineered matrix has not been systematically investigated. Here, we developed an in vitro 3D engineered CRC (3D-eCRC) tissue model using the natural-synthetic hybrid biomaterial PEG-fibrinogen and three CRC cell lines, HCT 116, HT-29, and SW480. To better recapitulate native tumor conditions, our 3D-eCRC model supported higher cell density encapsulation (20 × 106 cells/mL) and enabled longer term maintenance (29 days) as compared to previously reported in vitro CRC models. The 3D-eCRCs formed using each cell line demonstrated line-dependent differences in cellular and tissue properties, including cellular growth and morphology, cell subpopulations, cell size, cell granularity, migration patterns, tissue growth, gene expression, and tissue stiffness. Importantly, these differences were found to be most prominent from Day 22 to Day 29, thereby indicating the importance of long-term culture of engineered CRC tissues for recapitulation and investigation of mechanistic differences and drug response. Our 3D-eCRC tissue model showed high potential for supporting future in vitro comparative studies of disease progression, metastatic mechanisms, and anti-cancer drug candidate response in a CRC cell line-dependent manner.

Ratiometric Inclusion of Fibroblasts Promotes Both Castration-Resistant and Androgen-Dependent Tumorigenic Progression in Engineered Prostate Cancer Tissues.

To investigate the ratiometric role of fibroblasts in prostate cancer (PCa) progression, this work establishes a matrix-inclusive, 3D engineered prostate cancer tissue (EPCaT) model that enables direct coculture of neuroendocrine-variant castration-resistant (CPRC-ne) or androgen-dependent (ADPC) PCa cells with tumor-supporting stromal cell types. Results show that the inclusion of fibroblasts within CRPC-ne and ADPC EPCaTs drives PCa aggression through significant matrix remodeling and increased proliferative cell populations. Interestingly, this is observed to a much greater degree in EPCaTs formed with a small number of fibroblasts relative to the number of PCa cells. Fibroblast coculture also results in ADPC behavior more similar to the aggressive CRPC-ne condition, suggesting fibroblasts play a role in elevating PCa disease state and may contribute to the ADPC to CRPC-ne switch. Bulk transcriptomic analyses additionally elucidate fibroblast-driven enrichment of hallmark gene sets associated with tumorigenic progression. Finally, the EPCaT model clinical relevancy is probed through a comparison to the Cancer Genome Atlas (TCGA) PCa patient cohort; notably, similar gene set enrichment is observed between EPCaT models and the patient primary tumor transcriptome. Taken together, study results demonstrate the potential of the EPCaT model to serve as a PCa-mimetic tool in future therapeutic development efforts.

Differentiating Engineered Tissue Images and Experimental Factors to Classify Cardiomyocyte Content.

In this study, we used machine learning (ML) to classify the cardiomyocyte (CM) content on day 10 of the differentiation of human-induced pluripotent stem cell (hiPSC)-laden microspheroids using easily acquirable nondestructive phase-contrast images taken in the middle of differentiation and tunable experimental parameters. Scale-up suspension culture, use of engineered tissues to support stem cell differentiation, and CM production for improved control over cellular microenvironment in the suspension system need nondestructive methods to track engineered tissue development. The ability to couple images that capture experimenter perceived "good" or "bad" batches based on visualization at early differentiation time points with actual experimental outcomes in an unbiased way is a step toward building these methods. In recent years, ML techniques have been successfully applied to identify critical process parameters and use this information to build models that describe process outcomes in cell production and hiPSC differentiation. Building upon these successes, here, we utilize convolutional neural networks (CNNs) to build a binary classifier model for CM content on differentiation day 10 (dd10) for hiPSC-CMs. We consider two separate data sets as potential input features for the classification models. The first set includes phase-contrast images of microspheroid tissues taken on days 3 and 5 of the differentiation batches at different experimental conditions. The second set supplements the images with tunable experimental differentiation parameters, such as cell concentration and microspheroids' size. The CM content classes were sufficient and insufficient. The accuracy of the CNN classifier using images only was 63%. The addition of experimental features increased the accuracy to 85%, indicating the importance of tunable parameters in predicting CM content. Impact statement Machine learning approaches were used to predict the final cardiomyocyte (CM) content class (sufficient vs. insufficient) of engineered cardiac tissue microspheroids produced through suspension-based cardiac differentiation of human-induced pluripotent stem cell-laden engineered tissue microspheroids. The models used specified experimental features and data collected using nondestructive inexpensive methods, specifically phase-contrast images taken during the initial days of differentiation as inputs. The best model was a convolutional neural network trained using experimental features and differentiation day 5 images. It classified the CM content with 85% accuracy and replicated and formalized experimenter's visual intuition about differentiation outcomes by incorporating images from early time points.

Human Induced Pluripotent Stem Cell Encapsulation Geometry Impacts Three-Dimensional Developing Human Engineered Cardiac Tissue Functionality.

Cardiac tissue engineering has been working to alleviate the immense burden of cardiovascular disease for several decades. To improve cardiac tissue homogeneity and cardiomyocyte (CM) maturation, in this study, we investigated altering initial encapsulation geometry in a three-dimensional (3D) direct cardiac differentiation platform. Traditional engineered cardiac tissue production utilizes predifferentiated CMs to produce 3D cardiac tissue and often involves various cell selection and exogenous stimulation methods to promote CM maturation. Starting tissue formation directly with human induced pluripotent stem cells (hiPSCs), rather than predifferentiated CMs, simplifies the engineered cardiac tissue formation process, making it more applicable for widespread implementation and scale-up. In this study, hiPSCs were encapsulated in poly (ethylene glycol)-fibrinogen in three tissue geometries (disc-shaped microislands, squares, and rectangles) and subjected to established cardiac differentiation protocols. Resulting 3D engineered cardiac tissues (3D-ECTs) from each geometry displayed similar CM populations (∼65%) and gene expression over time. Notably, rectangular tissues displayed less tissue heterogeneity and suggested more advanced features of maturing CMs, including myofibrillar alignment and Z-line formation. In addition, rectangular tissue showed significantly higher anisotropic contractile properties compared to square and microisland tissues (MI 0.28 ± 0.03, SQ 0.35 ± 0.05, RT 0.79 ± 0.04). This study demonstrates a straightforward method for simplifying and improving 3D-ECT production without the use of exogenous mechanical or electrical pacing and has the potential to be utilized in bioprinting and drug testing applications. Impact statement Current methods for improving cardiac maturation postdifferentiation remain tedious and complex. In this study, we examined the impact of initial encapsulation geometry on improvement of three-dimensional engineered cardiac tissue (3D-ECT) production and postdifferentiation maturation for three tissue geometries, including disc-shaped microislands, squares, and rectangles. Notably, rectangular 3D-ECTs displayed less tissue heterogeneity and more advanced features of maturing cardiomyocytes, including myofibrillar alignment, Z-line formation, and anisotropic contractile properties, compared to microisland and square tissues. This study demonstrates an initial human induced pluripotent stem cell-encapsulated rectangular tissue geometry can improve cardiac maturation, rather than implementing cell selection or tedious postdifferentiation manipulation, including exogenous mechanical and/or electrical pacing.

Endothelial colony forming cell rolling and adhesion supported by peptide-grafted hydrogels.

The aim of this study was to investigate the ability of peptides and peptide combinations to support circulating endothelial colony forming cell (ECFC) rolling and adhesion under shear flow, informing biomaterial design in moving toward rapid cardiovascular device endothelialization. ECFCs have high proliferative capability and can differentiate into endothelial cells, making them a promising cell source for endothelialization. Both single peptides and peptide combinations designed to target integrins α4ß1 and α5ß1 were coupled to poly(ethylene glycol) hydrogels, and their performance was evaluated by monitoring velocity patterns during the ECFC rolling process, in addition to firm adhesion (capture). Tether percentage and velocity fluctuation, a parameter newly defined here, were found to be valuable in assessing cell rolling velocity patterns and when used in combination were able to predict cell capture. REDV-containing peptides binding integrin α4ß1 have been previously shown to reduce ECFC rolling velocity but not to support firm adhesion. This study finds that the performance of REDV-containing peptides in facilitating ECFC dynamic adhesion and capture can be improved by combination with α5ß1 integrin-binding peptides, which support ECFC static adhesion. Moreover, when similar in length, the peptide combinations may have synergistic effects in capturing ECFCs. With matching lengths, the peptide combinations including CRRETAWAC(cyclic)+REDV, P_RGDS+KSSP_REDV, and P_RGDS+P_REDV showed high values in both tether percentage and velocity fluctuation and improvement in ECFC capture compared to the single peptides at the shear rate of 20 s-1. These newly identified peptide combinations have the potential to be used as vascular device coatings to recruit ECFCs. STATEMENT OF SIGNIFICANCE: Restoration of functional endothelium following placement of stents and vascular grafts is critical for maintaining long-term patency. Endothelial colony forming cells (ECFCs) circulating in blood flow are a valuable cell source for rapid endothelialization. Here we identify and test novel peptides and peptide combinations that can potentially be used as coatings for vascular devices to support rolling and capture of ECFCs from flow. In addition to the widely used assessment of final ECFC adhesion, we also recorded the rolling process to quantitatively evaluate the interaction between ECFCs and the peptides, obtaining detailed performance of the peptides and gaining insight into effective capture molecule design. Peptide combinations targeting both integrin α4ß1 and integrin α5ß1 showed the highest percentages of ECFC capture.

Droplet Microfluidics-Based Fabrication of Monodisperse Poly(ethylene glycol)-Fibrinogen Breast Cancer Microspheres for Automated Drug Screening Applications.

Spheroidal cancer microtissues are highly advantageous for a wide range of biomedical applications, including high-throughput drug screening, multiplexed target validation, mechanistic investigation of tumor-extracellular matrix (ECM) interactions, among others. Current techniques for spheroidal tissue formation rely heavily on self-aggregation of single cancer cells and have substantial limitations in terms of cell-type-specific heterogeneities, uniformity, ease of production and handling, and most importantly, mimicking the complex native tumor microenvironmental conditions in simplistic models. These constraints can be overcome by using engineered tunable hydrogels that closely mimic the tumor ECM and elucidate pathologically relevant cell behavior, coupled with microfluidics-based high-throughput fabrication technologies to encapsulate cells and create cancer microtissues. In this study, we employ biosynthetic hybrid hydrogels composed of poly(ethylene glycol diacrylate) (PEGDA) covalently conjugated to natural protein (fibrinogen) (PEG-fibrinogen, PF) to create monodisperse microspheres encapsulating breast cancer cells for 3D culture and tumorigenic characterization. A previously developed droplet-based microfluidic system is used for rapid, facile, and reproducible fabrication of uniform cancer microspheres with either MCF7 or MDA-MB-231 (metastatic) breast cancer cells. Cancer cell-type-dependent variations in cell viability, metabolic activity, and 3D morphology, as well as microsphere stiffness, are quantified over time. Particularly, MCF7 cells grew as tight cellular clusters in the PF microspheres, characteristic of their epithelial morphology, while MDA-MB-231 cells displayed elongated and invasive morphology, characteristic of their mesenchymal and metastatic nature. Finally, the translational potential of the cancer microsphere platform toward high-throughput drug screening is also demonstrated. With high uniformity, scalability, and control over engineered microenvironments, the established cancer microsphere model can be potentially used for mechanistic studies, fabrication of modular cancer microtissues, and future drug-testing applications.

Diet as a Risk Factor for Early-Onset Colorectal Adenoma and Carcinoma: A Systematic Review.

Background: Colorectal cancer in adults 50 years old and younger is increasing in incidence worldwide. Diet may be a modifiable risk factor. The objective of this study was to examine evidence regarding the association between diet and the risk of developing early-onset colorectal cancer (EOCRC) and early-onset colorectal adenomas in young adults. Methods: PUBMED, Web of Science, and Embase were systematically searched for studies examining dietary intake as a risk factor for EOCRC and early-onset colorectal adenomas. Results were synthesized narratively due to the heterogeneity of the studies. Results: Of the 415 studies identified, ten met the inclusion criteria. Of these ten studies, four provided data on dietary risk factors for early-onset colorectal adenomas and six provided data on dietary risk factors for EOCRC. The four studies that measured colorectal adenoma occurrence reported an increased incidence with high sugar sweetened beverage intake, a higher pro-inflammatory diet, a higher Western diet score and higher sulfur microbial diet score. A protective effect against early-onset colorectal adenomas was observed in those who had a higher Prudent diet score or higher adherence to other health dietary approaches (Dietary Approaches to Stop Hypertension, Alternative Healthy Eating Index-2010, or the alternative Mediterranean diet). Those who consumed large amounts of deep-fried foods, refined foods, followed a high fat diet, consumed large amounts of sugary drinks and desserts, and had low folate and fiber consumption had a significantly higher occurrence of EOCRC. A protective effect against EOCRC was observed for those who consumed more fruits and vegetables, high amounts of micronutrients and those who adhered to a vegetarian diet. Conclusions: The results of this study reveal various dietary habits may be risk factors or protective against early-onset colorectal cancer and adenomas. Future research should focus on large prospective cohort studies with long-term follow-up to confirm published results and further examine whether differences in diet quality are associated with EOCRC risk.

Engineered colorectal cancer tissue recapitulates key attributes of a patient-derived xenograft tumor line.

The development of physiologically relevantin vitrocolorectal cancer (CRC) models is vital for advancing understanding of tumor biology. Although CRC patient-derived xenografts (PDXs) recapitulate key patient tumor characteristics and demonstrate high concordance with clinical outcomes, the use of thisin vivomodel is costly and low-throughput. Here we report the establishment and in-depth characterization of anin vitrotissue-engineered CRC model using PDX cells. To form the 3D engineered CRC-PDX (3D-eCRC-PDX) tissues, CRC PDX tumors were expandedin vivo, dissociated, and the isolated cells encapsulated within PEG-fibrinogen hydrogels. Following PEG-fibrinogen encapsulation, cells remain viable and proliferate within 3D-eCRC-PDX tissues. Tumor cell subpopulations, including human cancer and mouse stromal cells, are maintained in long-term culture (29 days); cellular subpopulations increase ratiometrically over time. The 3D-eCRC-PDX tissues mimic the mechanical stiffness of originating tumors. Extracellular matrix protein production by cells in the 3D-eCRC-PDX tissues resulted in approximately 57% of proteins observed in the CRC-PDX tumors also being present in the 3D-eCRC-PDX tissues on day 22. Furthermore, we show congruence in enriched gene ontology molecular functions and Hallmark gene sets in 3D-eCRC-PDX tissues and CRC-PDX tumors compared to normal colon tissue, while prognostic Kaplan-Meier plots for overall and relapse free survival did not reveal significant differences between CRC-PDX tumors and 3D-eCRC-PDX tissues. Our results demonstrate high batch-to-batch consistency and strong correlation between ourin vitrotissue-engineered PDX-CRC model and the originatingin vivoPDX tumors, providing a foundation for future studies of disease progression and tumorigenic mechanisms.

Consensus molecular subtype differences linking colon adenocarcinoma and obesity revealed by a cohort transcriptomic analysis.

Colorectal cancer (CRC) is the third-leading cause of cancer-related deaths in the United States and worldwide. Obesity-a worldwide public health concern-is a known risk factor for cancer including CRC. However, the mechanisms underlying the link between CRC and obesity have yet to be fully elucidated in part because of the molecular heterogeneity of CRC. We hypothesized that obesity modulates CRC in a consensus molecular subtype (CMS)-dependent manner. RNA-seq data and associated tumor and patient characteristics including body weight and height data for 232 patients were obtained from The Cancer Genomic Atlas-Colon Adenocarcinoma (TCGA-COAD) database. Tumor samples were classified into the four CMSs with the CMScaller R package; body mass index (BMI) was calculated and categorized as normal, overweight, and obese. We observed a significant difference in CMS categorization between BMI categories. Differentially expressed genes (DEGs) between obese and overweight samples and normal samples differed across the CMSs, and associated prognostic analyses indicated that the DEGs had differing associations on survival. Using Gene Set Enrichment Analysis, we found differences in Hallmark gene set enrichment between obese and overweight samples and normal samples across the CMSs. We constructed Protein-Protein Interaction networks and observed differences in obesity-regulated hub genes for each CMS. Finally, we analyzed and found differences in predicted drug sensitivity between obese and overweight samples and normal samples across the CMSs. Our findings support that obesity impacts the CRC tumor transcriptome in a CMS-specific manner. The possible associations reported here are preliminary and will require validation using in vitro and animal models to examine the CMS-dependence of the genes and pathways. Once validated the obesity-linked genes and pathways may represent new therapeutic targets to treat colon cancer in a CMS-dependent manner.

Tunable three-dimensional engineered prostate cancer tissues for in vitro recapitulation of heterogeneous in vivo prostate tumor stiffness.

In this manuscript we report the establishment and characterization of a three-dimensional in vitro, coculture engineered prostate cancer tissue (EPCaT) disease model based upon and informed by our characterization of in vivo prostate cancer (PCa) xenograft tumor stiffness. In prostate cancer, tissue stiffness is known to impact changes in gene and protein expression, alter therapeutic response, and be positively correlated with an aggressive clinical presentation. To inform an appropriate stiffness range for our in vitro model, PC-3 prostate tumor xenografts were established. Tissue stiffness ranged from 95 to 6,750 Pa. Notably, xenograft cell seeding density significantly impacted tumor stiffness; a two-fold increase in the number of seeded cells not only widened the tissue stiffness range throughout the tumor but also resulted in significant spatial heterogeneity. To fabricate our in vitro EPCaT model, PC-3 castration-resistant prostate cancer cells were co-encapsulated with BJ-5ta fibroblasts within a poly(ethylene glycol)-fibrinogen matrix augmented with excess poly(ethylene glycol)-diacrylate to modulate the matrix mechanical properties. Encapsulated cells temporally remodeled their in vitro microenvironment and enrichment of gene sets associated with tumorigenic progression was observed in response to increased matrix stiffness. Through variation of matrix composition and culture duration, EPCaTs were tuned to mimic the wide range of biomechanical cues provided to PCa cells in vivo; collectively, a range of 50 to 10,000 Pa was achievable. Markedly, this also encompasses published clinical PCa stiffness data. Overall, this study serves to introduce our bioinspired, tunable EPCaT model and provide the foundation for future PCa progression and drug development studies. STATEMENT OF SIGNIFICANCE: The development of cancer models that mimic the native tumor microenvironment (TME) complexities is critical to not only develop effective drugs but also enhance our understanding of disease progression. Here we establish and characterize our 3D in vitro engineered prostate cancer tissue model with tunable matrix stiffness, that is inspired by this study's spatial characterization of in vivo prostate tumor xenograft stiffness. Notably, our model's mimicry of the TME is further augmented by the inclusion of matrix remodeling fibroblasts to introduce cancer-stromal cell-cell interactions. This study addresses a critical unmet need in the field by elucidating the prostate tumor xenograft stiffness range and establishing a foundation for recapitulating the biomechanics of site-of-origin and soft tissue metastatic prostate tumors in vitro.

Engineered cardiac tissue microsphere production through direct differentiation of hydrogel-encapsulated human pluripotent stem cells.

Engineered cardiac tissues that can be directly produced from human induced pluripotent stem cells (hiPSCs) in scalable, suspension culture systems are needed to meet the demands of cardiac regenerative medicine. Here, we demonstrate successful production of functional cardiac tissue microspheres through direct differentiation of hydrogel encapsulated hiPSCs. To form the microspheres, hiPSCs were suspended within the photocrosslinkable biomaterial, PEG-fibrinogen (25 million cells/mL), and encapsulated at a rate of 420,000 cells/minute using a custom microfluidic system. Even at this high cell density and rapid production rate, high intra-batch and batch-to-batch reproducibility was achieved. Following microsphere formation, hiPSCs maintained high cell viability and continued to grow within and beyond the original PEG-fibrinogen matrix. These initially soft microspheres (<250 Pa) supported efficient cardiac differentiation; spontaneous contractions initiated by differentiation day 8, and the microspheres contained >75% cardiomyocytes (CMs). CMs responded appropriately to pharmacological stimuli and exhibited 1:1 capture up to 6.0 Hz when electrically paced. Over time, cells formed cell-cell junctions and aligned myofibril fibers; engineered cardiac microspheres were maintained in culture over 3 years. The capability to rapidly generate uniform cardiac microsphere tissues is critical for advancing downstream applications including biomanufacturing, multi-well plate drug screening, and injection-based regenerative therapies.

Cell engraftment, vascularization, and inflammation after treatment of equine distal limb wounds with endothelial colony forming cells encapsulated within hydrogel microspheres.

BACKGROUND: Endothelial colony forming cells (ECFCs) may be useful therapeutically in conditions with poor blood supply, such as distal limb wounds in the horse. Encapsulation of ECFCs into injectable hydrogel microspheres may ensure cell survival and cell localization to improve neovascularization and healing. Autologous ECFCs were isolated from 6 horses, labeled with quantum nanodots (QD), and a subset were encapsulated in poly(ethylene) glycol fibrinogen microspheres (PEG-Fb MS). Full-thickness dermal wounds were created on each distal limb and injected with empty PEG-Fb MS, serum, ECFCs, or ECFCs encapsulated into PEG- Fb MS (ECFC/MS). Analysis included wound surface area (WSA), granulation tissue scoring (GS), thermography, collagen density staining, and immunohistochemical staining for endothelial and inflammatory cells. The purpose of this study was to track cell location and evaluate wound vascularization and inflammatory response after injection of ECFC/MS or naked ECFCs in equine distal limb wounds. RESULTS: ECFCs were found near and within newly formed blood vessels up to 3 weeks after injection. ECFC and ECFC/MS groups had the greatest blood vessel quantity at week 1 in the wound periphery. Wounds treated with ECFCs and ECFC/MS had the lowest density of neutrophils and macrophages at week 4. There were no significant effects of ECFC or ECFC/MS treatment on other measured parameters. CONCLUSIONS: Injection of microsphere encapsulated ECFCs was practical for clinical use and well-tolerated. The positive ECFC treatment effects on blood vessel density and wound inflammation warrant further investigation.

Microfluidic Production of Cell-Laden Microspheroidal Hydrogels with Different Geometric Shapes.

Providing control over the geometric shape of cell-laden hydrogel microspheroids, such as diameter and axial ratio, is critical for their use in biomedical applications. Building on our previous work establishing a microfluidic platform for production of large cell-laden microspheres, here we establish the ability to produce microspheroids with varying axial ratio (microrods) and elucidate the mechanisms controlling microspheroidal geometry. Microspheroids with radial diameters ranging from 300 to over 1000 µm and axial ratios from 1.3 to 3.6 were produced. Although for microfluidic devices with small channel sizes (typically <500 µm) the mechanisms governing geometric control have been investigated, these relationships were not directly translatable to production of larger microspheroids (radial diameter 102 - 103 µm) in microfluidic devices with larger channel sizes (up to 1000 µm). In particular as channel size was increased, fluid density differences became more influential in geometric control. We found that two parameters, narrowing ratio (junction diameter over outlet diameter) and flow fraction (discrete phase flow rate over total flow rate), were critical in adjusting the capillary number, modulation of which has been previously shown to enable control over microspheroid diameter and axial ratio. By changing the device design and the experimental conditions, we exploited the relationship between these parameters to predictably modulate microspheroid geometric shape. Finally, we demonstrated the applicability to tissue engineering through encapsulation of fibroblasts and endothelial colony forming cells (ECFCs) in hydrogel microspheroids with different axial ratios and negligible loss of cell viability. This study advances microfluidic production of large cell-laden microspheroids (microspheres and microrods) with controllable size and geometry, opening the door for further investigation of geometric shape-related biomedical applications such as engineered tissue formation.

Rapid Production of Cell-Laden Microspheres Using a Flexible Microfluidic Encapsulation Platform.

This study establishes a novel microfluidic platform for rapid encapsulation of cells at high densities in photocrosslinkable microspherical hydrogels including poly(ethylene glycol)-diacrylate, poly(ethylene glycol)-fibrinogen, and gelatin methacrylate. Cell-laden hydrogel microspheres are advantageous for many applications from drug screening to regenerative medicine. Employing microfluidic systems is considered the most efficient method for scale-up production of uniform microspheres. However, existing platforms have been constrained by traditional microfabrication techniques for device fabrication, restricting microsphere diameter to below 200 µm and making iterative design changes time-consuming and costly. Using a new molding technique, the microfluidic device employs a modified T-junction design with readily adjustable channel sizes, enabling production of highly uniform microspheres with cell densities (10-60 million cells mL-1 ) and a wide range of diameters (300-1100 µm), which are critical for realizing downstream applications, through rapid photocrosslinking (≈1 s per microsphere). Multiple cell types are encapsulated at rates of up to 1 million cells per min, are evenly distributed throughout the microspheres, and maintain high viability and appropriate cellular activities in long-term culture. This microfluidic encapsulation platform is a valuable and readily adoptable tool for numerous applications, including supporting injectable cell therapy, bioreactor-based cell expansion and differentiation, and high throughput tissue sphere-based drug testing assays.

Growth and function of equine endothelial colony forming cells labeled with semiconductor quantum dots.

BACKGROUND: Endothelial progenitor cells (EPCs) contribute to neovascularization and vascular repair in vivo and are attractive for clinical use in ischemic disease. Tracking of stem and progenitor cells is essential to determine engraftment after administration. Semiconductor quantum dots (QD) are promising for cell labeling due to their ease of uptake by many cell lines and their continued presence after many cell generations. The purpose of this study was to evaluate function and growth of equine EPCs after QD labeling. Additionally, this study evaluated the duration of QD label retention and mechanisms of QD label loss. RESULTS: Endothelial colony forming cells (ECFCs) from adult horses (N = 3) were employed for this study, with QD labeled and unlabeled ECFCs tested from each horse. Cell proliferation of ECFCs labeled with QD at 20 nM was quantified by comparing the number of cell doublings per day (NCD) and the population doubling time (PDT) in labeled and unlabeled cells. Function of labeled and unlabeled ECFCs was assessed by comparing uptake of acetylated low-density lipoprotein (DiO-Ac-LDL) and tubule formation on growth factor containing matrix. Cell proliferation was not impacted by QD labeling; both NCD (p = 0. 95) and PDT (P = 0. 91) did not differ between unlabeled and QD labeled cells. Function of ECFCs assessed by DiO-Ac-LDL and tubule formation was also not different between unlabeled and QD labeled cells (P = 0. 33 and P = 0. 52, respectively). ECFCs retained their QD labeling over 7 passages with both 5 nM and 20 nM label concentrations. Reduction in label intensity was observed over time, and the mechanism was determined to be cell division. CONCLUSIONS: Equine ECFCs are effectively labeled with QD, and QD concentrations up to 20 nM do not affect cell growth or function. QD label loss is a result of cell division. The use of QD labeling with equine EPCs may be an ideal way to track engraftment of EPCs for in vivo applications.

A Microvascularized Tumor-mimetic Platform for Assessing Anti-cancer Drug Efficacy.

Assessment of anti-cancer drug efficacy in in vitro three-dimensional (3D) bioengineered cancer models provides important contextual and relevant information towards pre-clinical translation of potential drug candidates. However, currently established models fail to sufficiently recapitulate complex tumor heterogeneity. Here we present a chip-based tumor-mimetic platform incorporating a 3D in vitro breast cancer model with a tumor-mimetic microvascular network, replicating the pathophysiological architecture of native vascularized breast tumors. The microfluidic platform facilitated formation of mature, lumenized and flow-aligned endothelium under physiological flow recapitulating both high and low perfused tumor regions. Metastatic and non-metastatic breast cancer cells were maintained in long-term 3D co-culture with stromal fibroblasts in a poly(ethylene glycol)-fibrinogen hydrogel matrix within adjoining tissue chambers. The interstitial space between the chambers and endothelium contained pores to mimic the "leaky" vasculature found in vivo and facilitate cancer cell-endothelial cell communication. Microvascular pattern-dependent flow variations induced concentration gradients within the 3D tumor mass, leading to morphological tumor heterogeneity. Anti-cancer drugs displayed cell type- and flow pattern-dependent effects on cancer cell viability, viable tumor area and associated endothelial cytotoxicity. Overall, the developed microfluidic tumor-mimetic platform facilitates investigation of cancer-stromal-endothelial interactions and highlights the role of a fluidic, tumor-mimetic vascular network on anti-cancer drug delivery and efficacy for improved translation towards pre-clinical studies.

Testicular germ line cell identification, isolation, and transplantation in two North American catfish species.

Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15 µm, diameter with distinct nucleus 7-8 µm diameter) and spermatogonia (12-15 µm, with distinct nucleus 6-7.5 µm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15 µm) and type B spermatogonia (diameter 10-11 µm). Germ cells were confirmed with expression of vasa. Blastula-stage embryos of channel catfish (I. punctatus) were injected with freshly dissociated blue catfish testicular germ cells as donor cells for transplantation. Seventeen days after the transplantation, 33.3% of the triploid channel catfish fry were determined to be xenogenic catfish. This transplantation technique was efficient, and these xenogenic channel catfish need to be grown to maturity to verify their reproductive capacity and to verify that for the first time SSCs injected into blastulae were able to migrate to the genital ridge and colonize. These results open the possibility of artificially producing xenogenic channel catfish males that can produce blue catfish sperm and mate with normal channel catfish females naturally. The progeny would be all C × B hybrid catfish, and the efficiency of hybrid catfish production could be improved tremendously in the catfish industry.

Encapsulation of Equine Endothelial Colony Forming Cells in Highly Uniform, Injectable Hydrogel Microspheres for Local Cell Delivery.

A common challenge in cell therapy is the inability to routinely maintain survival and localization of injected therapeutic cells. Delivering cells by direct injection increases the flexibility of clinical applications, but may cause low cell viability and retention rates due to the high shear forces in the needle and mechanical wash out. In this study, we encapsulated endothelial colony forming cells (ECFCs) in poly(ethylene glycol)-fibrinogen (PF) hydrogel microspheres using a custom-built microfluidic device; this system supports rapid encapsulation of high cell concentrations (10 million cells per mL) and resulting cell-laden microspheres are highly uniform in shape and size. The encapsulated ECFCs were shown to have >95% viability and continued to rapidly proliferate. Expression of cell markers (von Willebrand factor, CD105, and CD14), the ability to form tubules on basement membrane matrix, and the ability to take up low-density lipoprotein were similar between pre- and post-encapsulated cells. Viability of encapsulated ECFCs was maintained after shear through 18-23-gauge needles. Ex vivo and in vivo cell delivery studies were performed by encapsulating and injecting autologous equine ECFCs subcutaneously into distal limb full-thickness wounds of adult horses. Injected ECFCs were visualized by labeling with fluorescent nanodots before encapsulation. One week after injection, confocal microscopy analysis of biopsies of the leading edges of the wounds showed that the encapsulated ECFCs migrated into the surrounding host tissue indicating successful retention and survival of the delivered ECFCs. Rapid, scalable cell encapsulation into PF microspheres was demonstrated to be practical for use in large animal cell therapy and is a clinically relevant method to maintain cell retention and survival after local injection.

PEG-fibrinogen hydrogels for three-dimensional breast cancer cell culture.

Tissue-engineered three-dimensional (3D) cancer models employing biomimetic hydrogels as cellular scaffolds provide contextual in vitro recapitulation of the native tumor microenvironment, thereby improving their relevance for use in cancer research. This study reports the use of poly(ethylene glycol)-fibrinogen (PF) as a suitable biosynthetic hydrogel for the 3D culture of three breast cancer cell lines: MCF7, SK-BR-3, and MDA-MB-231. Modification of the matrix characteristics of PF hydrogels was achieved by addition of excess poly(ethylene glycol) diacrylate, which resulted in differences in Young's moduli, degradation behavior, release kinetics, and ultrastructural variations in scaffold microarchitecture. Cancer cells were maintained in 3D culture with high viability within these hydrogels and resulted in cell-type dependent morphological changes over time. Cell proliferation and 3D morphology within the hydrogels were visualized through immunofluorescence staining. Finally, spatial heterogeneity of colony area within the hydrogels was quantified, with peripheral cells forming colonies of higher area compared to those in the interior regions. Overall, PF-based hydrogels facilitate 3D culture of breast cancer cells and investigation of cellular behavior in response to varying matrix characteristics. PF-based cancer models could be potentially used in future investigations of cancer biology and in anti-cancer drug-testing applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 236-252, 2017.