Alginate core–shell microcapsule reduces the DMSO addition-induced osmotic damage to cells by inhibiting cellular blebs

In cryopreservation, the addition of cryoprotectant can change the intra- and extra-cellular osmotic pressure, affect the cell morphology, and induce blebs on the plasma membrane. In this study, the blebs of cells microencapsulated in the alginate microsphere induced by osmotic shock were studied, and the effects of microencapsulation on bleb size and cell viability were determined. Firstly, a coaxial co-flow focusing device was applied to generate cell-laden microcapsules using alginate hydrogel in this paper. Then, cellular blebs induced by DMSO with various concentrations under microencapsulation were com-pared with that when non-encapsulated, and the dynamic process of cellular bleb was investigated. Finally, the qualitative relationship between bleb size and cell viability in the presence of DMSO was built, and thus the effects of microencapsulation on bleb size and viability were evaluated. The results show that the bleb size is smaller and the cell viability is higher, and cell microencapsulation can signif-icantly inhibit the excessively large blebs generated on the cell membrane and reduce the osmotic dam-age to cells when loading cryoprotectant and then to improve cell viability during cryopreservation. This work can provide insights for optimizing cryoprotectant-loading protocols, offer a new avenue to study cell blebbing, and advance future research on cryopreservation of rare cells and biomaterials.     

Dimethyl Sulfoxide-Free Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells Based on Zwitterionic Betaine and Electroporation

The effective cryopreservation of mesenchymal stem cells (MSCs) is indispensable to the operation of basic research and clinical transplantation. The prevalent protocols for MSC cryopreservation utilize dimethyl sulfoxide (DMSO), which is easily permeable and able to protect MSCs from cryo-injuries, as a primary cryoprotectant (CPA). However, its intrinsic toxicity and adverse effects on cell function remain the bottleneck of MSC cryopreservation. In this work, we cryopreserved human umbilical cord mesenchymal stem cells (UCMSCs) using zwitterionic betaine combined with electroporation without any addition of DMSO. Betaine was characterized by excellent compatibility and cryoprotective properties to depress the freezing point of pure water and balance the cellular osmotic stress. Electroporation was introduced to achieve intracellular delivery of betaine, intending to further provide comprehensive cryoprotection on UCMSCs. Compared with DMSO cryopreservation, UCMSCs recovered from the protocol we developed maintained the normal viability and functions and reduced the level of reactive oxygen species (ROS) that are harmful to cell metabolism. Moreover, the in vivo distribution of thawed UCMSCs was consistent with that of fresh cells monitored by a bioluminescence imaging (BLI) system. This work opens a new window of opportunity for DMSO-free MSC cryopreservation using zwitterionic compounds like betaine combined with electroporation.     

Three‐dimensional hydrogel encapsulated embryonic stem and carcinoma cells as culture platforms for cytotoxicity studies

The utility of alginate hydrogels for three‐dimensional (3‐D) culture of mouse embryonic stem cells (mESCs) and future development of 3‐D stem cell culture‐based in vitro screens of toxicity is described. Using alginate hydrogels of various stiffness, we first evaluated the impact of substrate modulus on mESC viability, proliferation, as well as expression of pluripotency and germ‐layer markers and observed that low concentration alginate hydrogels (0.5% and 1% alginate) were most suitable for long‐term culture of mESCs. These results were not unique to mESCs; long‐term viability and proliferation of mouse embryonic carcinoma cells (mECCs) was also best supported by similar conditions. Finally, we determined cytotoxic responses of alginate encapsulated cells to commercially available chemicals and interestingly observed similar responses for mESCs and mECCs, thereby suggesting that mECCs can predict stem cell responses to chemicals. These studies will facilitate future design of optimal stem cell‐based platforms of organ‐specific and developmental toxicity. © 2015 American Institute of Chemical Engineers AIChE J, 61: 3180–3184, 2015     

Bioinspired glycosaminoglycan hydrogels via click chemistry for 3D dynamic cell encapsulation

Cell encapsulation within 3D hydrogels is an attractive approach to develop effective cell-based therapies. However, little is known about how cells respond to the dynamic microenvironment resulting from hydrogel gelation-based cell encapsulation. Here, a tunable biomimetic hydrogel system that possesses alterable gelation kinetics and biologically relevant matrix stiffness is developed to study 3D dynamic cellular responses during encapsulation. Hydrogels are synthesized by crosslinking thiolated hyaluronic acid and thiolated chondroitin sulfate with poly(ethylene glycol) diacrylate under cell-compatible conditions. Hydrogel properties are tailored by altering thiol substitution degrees of glycosaminoglycans or molecular weights of crosslinkers. Encapsulation of human mesenchymal stem cells through hydrogel gelation reveals high cell viability as well as a three-stage gelation-dependent cellular response in real-time focal adhesion kinase (FAK) phosphorylation in live single cells. Furthermore, stiffer hydrogels result in higher equilibrium FAK activity and enhanced actin protrusions. Our results demonstrate the promise of hydrogel-mediated cellular responses during cell encapsulation. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47212.     

Investigation of encapsulation of pancreatic beta cells and curcumin within alginate microcapsules

Cell encapsulation is an ideal approach for the replacement of pancreatic function in Type 1 diabetes. Poor biocompatibility of microcapsules generates an inflammatory response in the implantation site and induces fibrosis infiltration, which causes microencapsulated cell death and graft failure. To prevent inflammation after implantation, composite microcapsules that exhibit anti-inflammatory properties were designed. This study is about encapsulating beta cells and curcumin within 1.5% alginate by the jet-breaking regime of the syringe pump. The microcapsules’ size distribution and rate of the alginate solution were characterized to find uniform particles. Micro-size particles were attained at a rate of 25 mL/min. Uniform spherical microcapsules (200–300 μm) were created in large amounts in a short period. Microcapsule breakage was less than 3% during 7 days, which demonstrated the stability of the encapsulation method. Insulin secretion and cell viability assays were performed 1, 3, and 7 days after microencapsulation by glucose-stimulated insulin secretion (GSIS) and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays. No significant differences in the amount of insulin secretion and beta cell viability were observed among free cells, alginate microcapsules, and curcumin-alginate microcapsules during 7 days (p > 0.05). Therefore, the curcumin and alginate membrane did not show any harmful impacts on the function and survival of the beta cells.     

Differential Responses to Bioink-Induced Oxidative Stress in Endothelial Cells and Fibroblasts

A hydrogel system based on oxidized alginate covalently crosslinked with gelatin (ADA-GEL) has been utilized for different biofabrication approaches to design constructs, in which cell growth, proliferation and migration have been observed. However, cell–bioink interactions are not completely understood and the potential effects of free aldehyde groups on the living cells have not been investigated. In this study, alginate, ADA and ADA-GEL were characterized via FTIR and NMR, and their effect on cell viability was investigated. In the tested cell lines, there was a concentration-dependent effect of oxidation degree on cell viability, with the strongest cytotoxicity observed after 72 h of culture. Subsequently, primary human cells, namely fibroblasts and endothelial cells (ECs) were grown in ADA and ADA-GEL hydrogels to investigate the molecular effects of oxidized material. In ADA, an extremely strong ROS generation resulting in a rapid depletion of cellular thiols was observed in ECs, leading to rapid necrotic cell death. In contrast, less pronounced cytotoxic effects of ADA were noted on human fibroblasts. Human fibroblasts had higher cellular thiol content than primary ECs and entered apoptosis under strong oxidative stress. The presence of gelatin in the hydrogel improved the primary cell survival, likely by reducing the oxidative stress via binding to the CHO groups. Consequently, ADA-GEL was better tolerated than ADA alone. Fibroblasts were able to survive the oxidative stress in ADA-GEL and re-entered the proliferative phase. To the best of our knowledge, this is the first report that shows in detail the relationship between oxidative stress-induced intracellular processes and alginate di-aldehyde-based bioinks.     

大孔型纤维素硫酸钠-聚二甲基二烯丙基氯化铵微胶囊固定假丝酵母产脂肪酶

脂肪酶（1ipase,EC．3．1．1．3）能够分解或合成甘油酸酯键．近年来,随着非水相酶学研究的进展,大大扩展了脂肪酶在有机合成、外消旋体拆分等方面的用途．到目前,有关游离及固体培养产脂肪酶的方法已有较多的报道．固定化技术由于在提高产品产率和简化下游分离过程等方面特殊的优越性,也被应用于固定化发酵胞外酶．     

Oxygen consumption and diffusion in assemblages of respiring spheres: Performance enhancement of a bioartificial pancreas

Microencapsulation provides an immune barrier for transplanted islets of Langerhans to treat diabetes, but it imposes oxygen diffusional limitations that can result in loss of viability and function. We investigate two methods to reduce oxygen transport limitations: (1) enhancement of the encapsulant oxygen permeability, for example, by combination of a highly concentrated perfluorocarbon (PFC) emulsion with alginate (PFC alginate); and (2) reduction of islet tissue size, for example, by dispersing the islets into single cells followed by reaggregation into cell clusters smaller than the original islet. A theoretical reaction-diffusion model is used to predict the three-dimensional distribution of oxygen partial pressure in a spherical microcapsule and a planar slab containing islet tissue, from which the loss of cell viability and the reduction in insulin secretion rate is estimated. Numerical simulations are carried out for normal alginate and PFC alginate to examine the effect of surface oxygen partial pressure, capsule diameter, slab thickness, and the size and density of dispersed islet tissue. Results show that hypoxic conditions can be reduced, thereby enhancing islet viability and substantially maintaining insulin secretion rate when PFC emulsion is incorporated in the encapsulation material or when smaller islet cell aggregates are used in both types of geometries.     

超薄壁结构海藻酸钙胶囊膜制备及其功能化研究新进展

海藻酸钙胶囊膜由于具有制备过程温和环保、材料生物相容性优良等优点,广泛应用于生物医药等领域。薄壁结构的胶囊膜可减小跨膜传质阻力,加速囊膜内外物质的交换,因而备受学术界和工业界的广泛关注。近年来,具有超薄壁结构的海藻酸钙胶囊膜的制备与改性成为一个研究热点。本文综述了超薄壁结构海藻酸钙胶囊膜的制备方法及其功能化的研究新进展,重点介绍了利用共挤出毛细管装置制备超薄壁结构的海藻酸钙胶囊膜、利用精蛋白吸附与仿生硅化技术对超薄壁结构海藻酸钙胶囊膜的有机/无机杂化处理,以及利用复合纳米响应性凝胶颗粒的方法和接枝响应性聚合物高分子的方法实现超薄壁结构海藻酸钙胶囊膜的功能化改性等方面的研究现状。     

自组装多肽水凝胶支架中MSCs的三维培养及成骨分化

使用一种新型人工设计自组装多肽(RADA16)水凝胶作为三维培养支架评价MSCs成骨分化情况。将人骨髓MSCs培养增殖后接种到水凝胶中,在成骨分化培养液中进一步培养1～3周。荧光染色法观察细胞形态和存活情况;组织学染色检测MSCs ALP活性;半定量RT-PCR分析成骨特异性基因的表达。绝大多数MSCs在水凝胶支架内能够存活,呈纺锤样形态。诱导培养后蛋白和基因表达水平均检测到ALP活性,在14天时达到峰值。骨晚期分化特异性基因BSP也有表达,且表达量随培养时间延长而增多。自组装多肽水凝胶为MSCs的黏附生长及向成骨细胞分化提供良好的三维微环境,有望成为极具吸引力的骨组织工程支架材料。     

Coated alginate microspheres: Factors influencing the controlled delivery of macromolecules

A systematic study of the mild alginate/polycation microencapsulation process, as applied to encapsulation of bioactive macromolecules such as proteins, was conducted. When protein drugs were suspended in sodium alginate solution and sprayed into 1.3% buffered calcium chloride to form cross-linked microcapsules, large (up to 90%) losses of encapsulation species were encountered, and moderate to strong protein-alginate interactions caused poor formation of capsules. As a result, a diffusion-filling technique was adopted in which blank alginate beads, coated twice with small amounts of polycation, were formed prior to drug loading. Protein was then loaded into these capsules by stepwise diffusion from solutions of increasing drug concentration. The drug-loaded capsules were coated with a final layer of polycation. In all, three polycation coatings were used, two prior to filling and one after filling. The first coating strongly influenced the size, integrity, and loading capacity of the capsules. Low concentrations of polycation resulted in poorly formed capsules with very low retention of the drug in the final capsule, while very high concentrations prevented the drug from entering the capsule at the filling stage. This first coat also affected the duration of drug release from the capsule and the size of the burst effect. The second coat had less effect on the capsule integrity, but it did influence the drug payload and relase profile. The final, sealing-coat had little effect on drug payload and only limited effect on the release profile up to a critical concentration, above which the release profile was not affected. For all coats, increasing polycation concentration decreased the burst effect, and caused the release profile to be more sustained. Encapsulation of a series of dextrans with increasing molecular weight revealed that the release profile was directly related to the molecular weight of the diffusing species, which was more sustained as molecular weight increased. We have shown that the choice of coating parameters in the diffusion-filled, alginate/polycation system is critical for successful drug delivery from these capsules. By carefully choosing the coating parameters, both the drug payload and the release profile can be fine-tuned to meet the desired profile.     

Encapsulation of anthocyanin with gelatin-alginate carrier by a specially designed ultrasonic encapsulator with adjustable frequency

Anthocyanins are of increasing interest for food applications due to their health benefits. However, their poor stability and susceptibility to unfavorable environmental conditions may limit their application. Microencapsulation offers enhanced stability and controlled release properties. The aim was designing an ultrasonic encapsulator with a variable frequency ultrasonic driver to encapsulate black carrot and mahaleb anthocyanins. First, a double-channel variable frequency and power adjustable ultrasonic driver and atomizer head were designed. The cooled gelation technique was used to encapsulate anthocyanin in an alginate-gelatin complex. Freeze-drying and air-drying techniques were used for drying of microcapsules. Encapsulation parameters were atomization frequency, alginate, and anthocyanin concentrations. The capsules were characterized by encapsulation efficiency, particle diameter, and controlled release rates. Controlled release tests were performed at two different temperatures and pH values. Encapsulation efficiency was 87%–95%, mean diameters were between 69 and 95 μm. Maximum release rates were obtained from freeze-dried mahaleb anthocyanin capsules. Release rates increased with increasing temperature and pH values. In addition, the experimental results showed that the increase in the amount of alginate used in encapsulation decreased the release rate. Moreover, the results proved that the designed encapsulator can be used for encapsulation in fields such as food, pharmaceuticals, polymers, chemistry, and materials.     

Cryopreservation of Mesenchymal Stem Cells Using Medical Grade Ice Nucleation Inducer

Mesenchymal stem cells (MSCs) can differentiate into multiple different tissue lineages and have favourable immunogenic potential making them an attractive prospect for regenerative medicine. As an essential part of the manufacturing process, preservation of these cells whilst maintaining potential is of critical importance. An uncontrolled area of storage remains the rate of change of temperature during freezing and thawing. Controlled-rate freezers attempted to rectify this; however, the change of phase from liquid to solid introduces two extreme phenomena; a rapid rise and a rapid fall in temperature in addition to the intended cooling rate (normally −1 °C/min) as a part of the supercooling event in cryopreservation. Nucleation events are well known to initiate the freezing transition although their active use in the form of ice nucleation devices (IND) are in their infancy in cryopreservation. This study sought to better understand the effects of ice nucleation and its active instigation with the use of an IND in both a standard cryotube with MSCs in suspension and a high-throughput adhered MSC 96-well plate set-up. A potential threshold nucleation temperature for best recovery of dental pulp MSCs may occur around −10 °C and for larger volume cell storage, IND and fast thaw creates the most stable process. For adhered cells, an IND with a slow thaw enables greatest metabolic activity post-thaw. This demonstrates a necessity for a medical grade IND to be used in future regenerative medicine manufacturing with the parameters discussed in this study to create stable products for clinical cellular therapies.     

3D Bioprinting of Human Adipose-Derived Stem Cells and Their Tenogenic Differentiation in Clinical-Grade Medium

Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 × 10⁶ ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-β3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE.     

Fabrication and characterization of natural origin chitosan‐ gelatin‐alginate composite scaffold by foaming method without using surfactant

A natural origin tripolymer scaffold from chitosan, gelatin, and alginate was fabricated by applying foaming method without adding any foam stabilizing surfactant. Previously, in foaming method of scaffold fabrication, toxic surfactants were used to stabilize the foam, but in this work, the use of surfactant has been avoided strictly, which can provide better environment for cellular response and viability. In foaming method, stable foam is produced simply by agitating the polymer (alginate‐gelatin) solution, and the foam is crosslinked with CaCl₂, glutaraldehyde, and chitosan to produce tripolymer alginate‐gelatin‐chitosan composite scaffold. Microscopic images of the composite scaffold revealed the presence of interconnected pores, mostly spread over the entire surface of the scaffold. The scaffold has a porosity of 90% with a mean pore size of 57 μm. Swelling and degradation studies of the scaffold showed that the scaffold possesses excellent properties of hydrophilicity and biodegradability. In vitro cell culture studies by seeding L929 mouse fibroblast cells on scaffold revealed excellent cell viability, proliferation rate and adhesion as indicated by MTT assay, DNA quantification, and phase contrast microscopy of cell‐scaffold construct. The natural origin composite scaffold fabricated by the simplest method i.e., foaming method, but without adding any surfactant, is cheap, biocompatible, and it might find potential applications in the field of tissue engineering. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Osteogenic Potential of Mesenchymal Stem Cells from Adipose Tissue,Bone Marrow and Hair Follicle Outer Root Sheath in a 3D Crosslinked Gelatin-Based Hydrogel

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy.     

Protective Effect of Encapsulation in Fermentation of Limonene-contained Media and Orange Peel Hydrolyzate

This work deals with the application of encapsulation technology to eliminate inhibition by D-limonene in fermentation of orange wastes to ethanol. Orange peel was enzymatically hydrolyzed with cellulase and pectinase. However, fermentation of the released sugars in this hydrolyzate by freely suspended S. cerevisiae failed due to inhibition by limonene. On the other hand, encapsulation of S. cerevisiae in alginate membranes was a powerful tool to overcome the negative effects of limonene. The encapsulated cells were able to ferment the orange peel hydrolyzate in 7 h, and produce ethanol with a yield of 0.44 g/g fermentable sugars. Cultivation of the encapsulated yeast in defined medium was successful, even in the presence of 1.5% (v/v) limonene. The capsules’ membranes were selectively permeable to the sugars and the other nutrients, but not limonene. While 1% (v/v) limonene was present in the culture, its concentration inside the capsules was not more than 0.054% (v/v).     

Influence of folate conjugation on the cellular uptake degree of poly(allylamine hydrochloride) microcapsules

The microcapsules in drug delivery systems can prevent degradation of drugs and help to control the release rate. To enhance the targeted delivery effect of the microcapsules to cancer cells, some specific ligands such as folic acid (FA) are necessarily further conjugated. Herein, covalent poly(allylamine hydrochloride) (PAH) multilayers were fabricated on CaCO₃ microparticles under the cross‐linking of glutaraldehyde, which were further immobilized with different amount of FA molecules via the spacer of diamino terminated poly(ethylene glycol) (PEG). As a comparison study, four types of microcapsules, i.e., the PAH capsules, the PAH capsules grafted with PEG, and the PAH capsules conjugated with two different amount of FA via the PEG spacer were prepared. Their chemical and physical structures were confirmed by infrared spectroscopy, UV–vis spectroscopy and scanning electron microscopy. In vitro cell culture found that the cellular uptake of the PAH capsules grafted with PEG was reduced significantly compared with that of the pure PAH capsules. The FA‐modified microcapsules could be selectively delivered into HepG2 tumor cells which overexpress FA receptors but not into the endothelial cells. The number of HepG2 cells which ingested the FA‐conjugated capsules showed a positive correlation with FA amount. The results indicate that these FA conjugated capsules have a high selectivity to be delivered to tumor cells, endowing them with a larger opportunity functioning as targeted delivery vehicle for anticancer drugs. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

The effect of selected parameters of formation on properties of alginate/Ca2+/oligochitosan capsules

Optimization of the technological parameters affecting the mechanical properties and permeability of capsules is essential to produce capsules with improved properties for cell immobilization. In the present paper, the effect of different parameters on the technological properties of alginate/Ca²⁺/oligochitosan capsules has been investigated. The correct adjustment of the alginate concentration in the polymer matrix and the oligochitosan molar mass, concentration and coating time, have been found to be key parameters in obtaining porous and mechanically stable alginate/Ca²⁺/oligochitosan capsules. Results showed that an increase in the coating time and concentration of the alginate generated more stable capsules with a reduced membrane cut‐off. Furthermore, we have established some correlations between capsule properties and the effectiveness of chitosan binding within the capsule's membrane. Data addressed herein could be a valid tool to fabricate optimized alginate/Ca²⁺/oligochitosan capsules with a potential for use in cell immobilization technology. Copyright © 2006 Society of Chemical Industry     

A magnetic resonance-compatible perfusion bioreactor system for three-dimensional human mesenchymal stem cell construct development

Human mesenchymal stem cells (hMSCs) have significant potential for therapeutic tissue regeneration and repair. The creation of functional 3D constructs from hMSCs depends on the innate ability of MSCs to proliferate and differentiate, and is strongly influenced by the culture conditions. An inherent challenge in investigating 3D cellular construct development is the dynamic monitoring of the cellular and physiological environment over the course of construct formation. In this project, a novel 3D MR-compatible perfusion bioreactor using 3D poly(ethylene terephthalate) scaffolds was developed to provide such monitoring. The bioreactor system integrates cell seeding and growth, supports high density 3D tissue construct growth and facilitates repeated nuclear magnetic resonance (MR) signal acquisitions under both static and perfusion conditions. The reactor system also has the capacity to modulate macroscopic flow modes that simulates various tissue growth environments with repeated MR signal acquisition, providing the ability to gain insight into the dynamic interplay between the stem cells in the developing constructs and their microenvironment. Using ¹H MR spectroscopy and MR imaging, localized spectroscopic data as well as imaging-based T₂ and diffusion quantification were acquired from the hMSC growth construct for up to 40 days.