Cell Attachment Efficiency Research Articles

Preparation of porous GelMA microcarriers by microfluidic technology for Stem-Cell culture

Gelatin methacrylamide porous microcarriers (GelMA PMS), one of the most promising three-dimensional cell culture scaffolds, are limited in large-scale applications due to lack of ideal preparation method, unclear regulation of pore size on cell behavior and a lack of research on the entire cell culture process. Herein, combining microfluidic synchronous photo-crosslinking and ice-templating method, a low-cost, easy-assembly and suitable for scalability method is proposed to prepare GelMA PMS for bone marrow mesenchymal stem cells (BMSCs) expansion. The pore size of GelMA PMS can be controlled by adjusting the freezing temperature (-20 ℃, −60 ℃, −196℃). As a result, PMS 60 (frozen at −60 ℃) with the optimized pore size of 25.3 ± 3.2 μm exhibited the highest cell attachment ratio (90.2%) and the largest cell spreading area (0.516 mm2/MS). More importantly, PMS 60 displayed higher cell attachment efficiency, proliferation rate, adipogenic, osteogenic, and chondrogenic differentiation potential than commercial microcarriers Cytodex-1. Furthermore, BMSCs not only achieved bead-to-bead transfer on PMS 60 but also exhibited potent proliferation rate after transfer to new blank microcarriers. Cultured BMSCs were efficiently harvested from PMS 60 without damaging cell viability. In addition, the cryopreservation and thawing process did not affect the PMS 60 to support cell proliferation, while cells loaded on Cytodex-1 could not continue to proliferate. Therefore, GelMA PMS fabricated in this work shows great potential application and provides insights for MSCs culture.

Effect of Dermal Fibroblast Conditioned Medium on Keratinocytes Irrespective of Age Group

Skin aging causes delayed re-epithelialisation and impaired wound healing. Thus, supplementation of wound healing mediators and extracellular matrix (ECM) components may be a potential treatment strategy for age-related impaired wound healing. Fibroblasts secrete wound-healing factors and can be collected from used medium, i.e., dermal fibroblast conditioned medium (DFCM). In this study, we elucidated the effect of DFCM on the in vitro wound healing of keratinocytes isolated from different age groups (≥18–35, 36–54, ≥55 years) via cell attachment, growth rate, and wound healing rate assays. The DFCM was prepared by culturing confluent fibroblasts with serum-free keratinocyte-specific (DFCM-KM) and fibroblast-specific (DFCM-FM) medium. The cell attachment efficiency decreased with the increase of age. However, keratinocyte attachment was enhanced in the DFCM-KM group, where it was 1.24, 1.27, and 1.32 times higher of cells concentration for the ≥18–35-, 36–54-, and ≥55-year age groups, respectively, as compared to the control group. The keratinocytes from each age group demonstrated a similar growth profile for all culture conditions, where the DFCM-KM group exhibited a comparable growth rate with the control group whilst the DFCM-FM group exhibited a significantly lower growth rate compared to the other groups. In contrast, the DFCM-FM group demonstrated a significantly higher healing rate in all age groups as compared to the DFCM-KM and control groups. However, there was no significant difference between the healing rates of the DFCM-KM and control groups. In conclusion, DFCM-KM enhanced keratinocyte attachment while DFCM-FM enhanced the keratinocyte healing rate irrespective of donor age, which indicated the potential application of DFCM in wound healing in aged skin.

Cell Response in Free-Packed Granular Systems.

The study of the interactions of living adherent cells with mechanically stable (visco)elastic materials enables understanding and exploitation of physiological phenomena mediated by cell-extracellular communication. Insights into the interaction of cells and surrounding objects with different stability patterns upon cell contact might unveil biological responses to engineer innovative applications. Here, we hypothesize that the efficiency of cell attachment, spreading, and movement across a free-packed granular bed of microparticles depends on the microparticle diameter, raising the possibility of a necessary minimum traction force for the reinforcement of cell-particle bonds and long-term cell adhesion. The results suggest that microparticles with diameters of 14-20 μm are prone to cell-mediated mobility, holding the potential of inducing early cell detachment, while objects with diameters from 38 to 85 μm enable long-lasting cell adhesion and proliferation. An in silico hybrid particle-based model that addresses the time-dependent biological mechanisms of cell adhesion is proposed, providing inspiration for engineering platforms to address healthcare-related challenges.

Comparison of mesenchymal stem cell attachment efficiency in acellular neural graft for peripheral nerve regeneration

Decellularized nerve allograft is an alternative to autologous nerve graft for nerve defects but has shown inferior clinical outcomes. Mesenchymal stem cells can play a key role in improving nerve regeneration of decellularized nerve allografts. The purpose of this study was to compare different mesenchymal stem cell seeding methods and to find the most efficient way to attach cells to nerve grafts for peripheral nerve regeneration. Wharton's jelly mesenchymal stem cells were collected from human umbilical cords and were seeded in the acellular nerve graft in five different ways as follows: PBS injection, fibrin glue drop, Matrigel drop, bioreactor, and Matrigel injection. A 6-mm sciatic nerve defect of Sprague–Dawley rats was bridged using mesenchymal stem cells-laden acellular nerve grafts according to the five seeding methods. Two days after implantation, the nerve tissue was biopsied and analyzed by the immunofluorescence staining of nuclei. The number of Wharton's jelly mesenchymal stem cells (+ h Nuclei) was counted in the inside, outside, and the total area of the graft sections under 200X magnification. The highest efficiency of mesenchymal stem cell attachment inside the graft and the highest total number of attached mesenchymal stem cells was observed in the group using Matrigel injection (p < 0.0001). This study showed mesenchymal stem cells can be more effectively attached to decellularized nerve graft using the injection method with Matrigel than other static or dynamic seeding methods in vivo.

Effect of Magnesium on Dentinogenesis of Human Dental Pulp Cells.

Introducing therapeutic ions into pulp capping materials has been considered a new approach for enhancing regeneration of dental tissues. However, no studies have been reported on its dentinogenic effects on human dental pulp cells (HDPCs). This study was designed to investigate the effects of magnesium (Mg2+) on cell attachment efficiency, proliferation, differentiation, and mineralization of HDPCs. HDPCs were cultured with 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM concentrations of supplemental Mg2+ and 0 mM (control). Cell attachment was measured at 4, 8, 12, 16, and 20 hours. Cell proliferation rate was evaluated at 3, 7, 10, 14, and 21 days. Crystal violet staining was used to determine cell attachment and proliferation rate. Alkaline phosphatase (ALP) activity was assessed using the fluorometric assay at 7, 10, and 14 days. Mineralization of cultures was measured by Alizarin red staining. Statistical analysis was done using multiway analysis of variance (multiway ANOVA) with Wilks' lambda test. Higher cell attachment was shown with 0.5 mM and 1 mM at 16 hours compared to control (P < 0.0001). Cells with 0.5 mM and 1 mM supplemental Mg2+ showed significantly higher proliferation rates than control at 7, 10, 14, and 21 days (P < 0.0001). However, cell proliferation rates decreased significantly with 4 mM and 8 mM supplemental Mg2+ at 14 and 21 days (P < 0.0001). Significantly higher levels of ALP activity and mineralization were observed in 0.5 mM, 1 mM, and 2 mM supplemental Mg2+ at 10 and 14 days (P < 0.0001). However, 8 mM supplemental Mg2+ showed lower ALP activity compared to control at 14 days (P < 0.0001), while 4 mM and 8 mM supplemental Mg2+showed less mineralization compared to control (P < 0.0001). The study indicated that the optimal (0.5–2 mM) supplemental Mg2+ concentrations significantly upregulated HDPCs by enhancing cell attachment, proliferation rate, ALP activity, and mineralization. Magnesium-containing biomaterials could be considered for a future novel dental pulp-capping additive in regenerative endodontics.

Dissolvable Gelatin-Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells.

Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells.

Reovirus σ1 Conformational Flexibility Modulates the Efficiency of Host Cell Attachment.

Reovirus attachment protein σ1 is a trimeric molecule containing tail, body, and head domains. During infection, σ1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows σ1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in σ1 from a compact form on virions to an extended form on ISVPs. To determine the importance of σ1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can cross-link σ1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that cross-link the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride, which blocks virion-to-ISVP conversion. Together, these data suggest that σ1 conformational flexibility modulates the efficiency of reovirus host cell attachment.IMPORTANCE Nonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, σ1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that cross-link σ1 monomers in nonreducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of σ1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that cross-linking the σ1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that σ1 conformational rearrangements mediate the efficiency of reovirus host cell binding.

Poly(Caprolactone)‐Poly(N‐Isopropyl Acrylamide)‐Fe3O4 Magnetic Nanofibrous Structure with Stimuli Responsive Drug Release

AbstractPoly(caprolactone; PCL)—poly(N‐isopropylacrylamie; PNIPAAm)—Fe3O4 fiber, that can be magnetically actuated, is reported. Here, a structure is engineered that can be utilized as a smart carrier for the release of chemotherapeutic drug via magneto‐thermal activation, with the aid of magnetic nanoparticles (MNPs). The magnetic measurement of the fibers revealed saturation magnetization values within the range of 1.2–2.2 emu g−1. The magnetic PCL‐PNIPAAm‐Fe3O4 scaffold shows a specific loss power value of 4.19 W g−1 at 20 wt% MNPs. A temperature increase of 40 °C led to a 600% swelling after only 3 h. Doxorubicin (DOX) as a model drug, demonstrates a controllable drug release profile. 39% ± 0.92 of the total drug loaded is released after 96 h at 37 °C, while 25% drug release in 3 h at 40 °C is detected. Cytotoxicity results show no significant difference in cell attachment efficiency between the MNP‐loaded fibers and control while the DOX‐loaded fibers effectively inhibited cell proliferation at 24 h matching the drug release profile. The noncytotoxic effect, coupled with the magneto‐thermal property and controlled drug release, renders excellent potential for these fibers to be used as a smart drug‐release agent for localized cancer therapy.

Fabrication of pH responsive coacervates using a polycation-b-polypropylene glycol diblock copolymer for versatile delivery platforms

A new poly (3-arginylamino propylene succinate-b-polypropylene glycol) (PAPS-PPG) diblock copolymer was synthesized by one-pot ring opening polymerization (ROP) technique. 1H NMR spectral technique was used to determine arginine conjugation efficiency and molecular weight of final polycations by using 4,4-dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA) as an internal standard. The PAAPS-PPG polycation was electrostatically complexed with heparin (HEP) at an isoelectric point to obtain PAAPS-PPG/HEP coacervate microparticles (Coa MPs). The PAAPS-PPG/HEP Coa MPs exhibit acid induced dissociation at pH 5, but PAAPS/HEP Coa MPs without PPG block do not show acid induced dissociation. The PPG block in PAAPS-PPG/HEP Coa MPs increased 20 folds of cell attachment efficiency on human mesenchymal stem cells (hMSCs). The PAAPS-PPG/HEP Coa MPs could effectively encapsulate siRNA and exhibit more than 95% cell viability up to 200μg/mL. Hence, the pH responsive PAAPS-PPG/HEP Coa MPs platform with enhanced stem cell attachment ability would find potential application in gene delivery.

3D MSC Culture in Saint-Gobain VueLife® FEP Bags Using Microcarriers

Background & Aim MSCs are anchorage-dependent cells that require interaction with a substrate for cell survival and active proliferation. Achieving clinically relevant autologous cell doses in vitro requires significant surface area. Microcarriers are a commonly used, high surface area 3D culture solution combined with traditional bioreactors for allogeneic therapies; however, bioreactors are operated at a much larger scale than that required for personalized medicine. Therefore, a solution combining mid-size, fully closed, single use bags from Saint-Gobain's VueLife® product line with commercially available microcarriers is promising for autologous scale expansion of MSCs. As demonstrated here, VueLife® FEP bags are particularly attractive for this application due to the inherent non-stick properties of the FEP, preventing cell and microcarrier adhesion to the bag surface. Methods, Results & Conclusion For this study, human bone marrow-derived MSCs were purchased from RoosterBio Inc. and cultured in RoosterNourish™ MSC-XF medium. To assess donor-dependent variability in the cell culture performance, three individual donors were compared. MSC expansion was performed in Saint-Gobain's VueLife® 32-C bags with Corning® CellBIND® polystyrene microcarriers and Corning® Synthemax™ II-coated dissolvable microcarriers. Protocols for MSC attachment, expansion and harvest were developed for both microcarrier systems. Cell expansion results after 6 days of culture using the microcarriers are summarized in Tables 1+2. Strong donor-dependent variability was observed in the outcome, with expansion results ranging from 4- to 21-fold increase. However, because the CellBIND® beads are not transparent, cell attachment and harvest efficiency cannot be easily tracked by microscopy and may have also impacted the outcome. Cell attachment and expansion was easier to observe on the dissolvable microcarriers, with clear attachment after four hours and full confluence after four days of culture (Figure 1). MSCs showed 24-26-fold expansion for the three donors (Table 2) using the Corning® dissolvable microcarriers. MSC surface marker expression levels were not affected by culture on either microcarrier system in VueLife® bags and comparable to expression in T-175 flasks, as shown by flow cytometry. To support further expansion towards clinically-relevant doses, additional beads and media may be added to the bags. In summary, MSC expansion can be facilitated using microcarriers in VueLife® FEP bags for patient specific therapies. Results of MSC expansion on Corning's CellBIND® and Synthemax™ II-coated microcarriers after 6 days in culture in Saint-Gobain VueLife® 32-C bags. Results are shown for three tested MSC donors (#164, #172 and #227).

An additive manufacturing approach to bioreactor design for mesenchymal stem cell culture

Bioreactor design is a challenging endeavour that aims to provide the most ideal environment in which cells can grow and biological reactions can occur. The emergence of regenerative medicine and stem cell therapies has led to the need for more diverse environmental requirements in the bioreactor design space.The study presented uses an additive manufacturing approach for the initial design phase of a packed/fluidized bed bioreactor for mesenchymal stem cell expansion. Combining 3D-printing with CFD for precision control over the bioreactor flow dynamics. Novel flow distributors were developed to engender swirling particle fluidization. The design was simulated and optimised using CFD, demonstrating an increase from 0.01 m/s to 0.02 m/s in the radial velocity of 3.0 mm macrocarriers (1080 kg/m3) at the minimum fluidization velocity. An autoclavable prototype was constructed to illustrate proof-of-concept in the use of swirling flow distribution to enhance cell attachment efficiency (compared to static culture system). Commercial Cytodex 1 carriers were tested: an improvement in attachment efficiency after 24 h from 50 % to 95 % was induced by the swirling flow distributor, with subsequent expansion of 2.4-fold after 6 days of culture.The computational design, modelling and 3D-printing of complex geometric architecture that control the flow dynamics within a bioreactor, provides a novel approach to bioprocess unit operation development for manufacturing novel ATMPs.

Suppression of time-dependent decay by controlling the redox balance of human induced pluripotent stem cells suspended in a cryopreservation solution

To scale-up a filling process in a cell manufacturing system, the impact of the processing time on the final product quality should be evaluated. It is known that the suspended cells in a cryopreservation solution are subjected to various types of stress factors, which eventually results in apoptotic or necrotic death. In this study, the aim was to investigate a critical factor of cell decay in human induced pluripotent stem cells suspended in a cryopreservation solution and to propose a methodology to maintain the viability of cells for a longer filling process time. Based on experimental results, it was determined that the accumulation of intracellular reactive oxygen species induces apoptosis of cells suspended in a cryopreservation solution. In addition, treatment with an antioxidant or a low-temperature condition was effective in suppressing the induction of apoptosis and subsequent cell death. In particular, the treatment at low temperature was able to maintain the attachment efficiency of suspended cells at a higher level compared to the treatment with antioxidants. The appropriate control of redox balance in cells suspended in a cryopreservation solution could improve the viability of cells in the filling process and lead to the successful lot size expansion of cell manufacturing systems.

Cytotoxic Effect of Chitosan Nanoparticles on Normal Human Dental Pulp Cells

Introduction: Recent in vitro studies have shown that chitosan nanoparticles could enhance the antimicrobial activity of several dental materials. However, the biocompatibility of these nanoparticles with normal human cells is still controversial. The aim of this study was to evaluate the potential toxicity of various sizes and concentrations of chitosan nanoparticles cultured with normal human dental pulp cells. Methods: Normal human dental pulp cells were derived from human dental pulp tissues and cultured with (50-67) nm and (318-350) nm chitosan nanoparticles in concentrations: 0.2 mg/mL, 0.5 mg/mL, 1 mg/mL, and 2 mg/mL as study groups, and 0 mg/mL as a control. The cell attachment efficiency for each group was assessed at 16 hours. The proliferation rate and cell viability were evaluated at days 7 and 14. Both, attachment efficiency and proliferation rate were assessed by measuring the optical density of crystal violet stained cells. The cell viability was determined by the activity of the mitochondrial dehydrogenase enzyme. Statistical analysis was performed using One-Way ANOVA and post hoc Tukey test. Results: All concentrations of the (50-67) nm group significantly reduced cell attachment efficiency in comparison with the control (p&lt;0.01) and with the (318-350) nm group (p&lt;0.01). All concentrations of both groups, (50-67) nm and (318-350) nm, significantly reduced cell proliferation and cell viability compared to the control in dose-dependent and size-associated manners. (p&lt;0.01). Conclusion: Chitosan nanoparticles exhibit a cytotoxic effect on normal human dental pulp cells

Inorganic Phosphate Effect of on Human Dental Pulp Cell Cultures

This study was designed to investigate the effect of inorganic phosphate (Pi) at different concentrations on odontogenesis of the normal human dental pulp cells (hDPCs). Normal human dental pulp cells derived from extracted pristine teeth were cultured in growth medium with supplements of inorganic phosphate (Pi) in 0 ppm, 2 ppm, 4 ppm, 5 ppm and 8 ppm, for the time intervals of 16 hours, 7, 14, and 21 days. Cell proliferation rates were measured by the optical density of crystal violet dye stained cells. ALP activity was measured by fluorometric assay. Expression of Dentin Sialoprotein (DSP) was measured by ELISA. The data were presented as the mean of triplicates. Statistical analysis was conducted using JMP Pro 12 (ver. 12.1.0) in one-way ANOVA and Tukey HSD post-hoc tests. Cell attachment efficiency was reduced significantly by additional Pi of 2, 4 and 5 ppm (P<0.05). At 21 days, cultures with 2, 4 and 5 ppm supplemental Pi displayed significantly higher cell proliferation rates compared to the control group at day 14 (P<0.05) and at day 21 (P<0.05). At day 7, cultures with 2, 4, 5 and 8 ppm supplemental Pi yield significantly higher levels of ALP activity (P<0.05) compared to the control group. At day 7, cultures with 5 ppm Pi supplement showed significantly higher levels of DSP expression (P<0.05) compared to the control group and the rest of the other groups. Supplemental Pi in concentration of 5 ppm could significantly induce proliferation and odontogenesis of hDPCs. This is the first report to demonstrate Pi-induced odontogenesis, leading to potential development and clinical application of future Pi containing dental pulp capping or root canal filling materials.

Collagen-Chitosan Hydrogel Formed in Situ Via Enzymatic Crosslinking

In this research, injectable collagen-chitosan conjugates (Col-Chit-Ph) hydrogel was formed by oxidative coupling of phenolic hydroxyl (Ph) groups in polymer chain via peroxidase-catalysed crosslinking reaction. The resulting Col-Chit-Ph solutions formed rapid hydrogel at physiological conditions by utilizing H2O2. The amount of chitosan in the compositions of Col-Chit-Ph hydrogels influenced the mechanical properties of the hydrogels. Here, conjugates having Col:Chit ratio of 1:1 showed greater resistance to compression compared to conjugates having Col:Chit ratio of 3:2. Meanwhile, the efficiency of cell attachment for conjugates with Col:Chit ratio of 4:1 is higher than conjugates with Col:Chit ratio of 1:1. Although the biological properties of hydrogel was good at Col:Chit ratio of 4:1, this hydrogel easily destroyed by external force. However, with higher amount of chitosan in the composite, hydrogels were more mechanically stable. This indicates the importance of the chitosan ratio to the novel Col-Chit-Ph injectable hydrogel composite.

Electrosprayed genipin cross-linked alginate-chitosan microcarriers for ex vivo expansion of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are potential therapeutic candidates, owing to their ability to differentiate into several cell types. However, the gap between availability and demand of MSCs requires alternative expansion methods from 2D culture flasks. Microcarriers are a promising approach for MSC expansion due to their high surface area-to-volume ratio. However, current commercial microcarriers do not provide the highest cell yield due to low cell attachment efficiencies and difficulty in cell detachment. This study developed a hydrogel-based microcarrier from genipin cross-linked alginate-chitosan beads. Alginate beads were produced by electrospraying before being coated with chitosan and cross-linked in genipin. The degree of cross-linking was determined through fluorescence reading of the genipin-chitosan conjugates. MSCs cultured on these microcarriers had a 26% higher cell attachment and twice the proliferation rate compared to the commercial microcarrier Cytodex 1. Cells easily detached from the hydrogel beads and did not require extended incubation periods or intense agitation during cell harvest. There was no significant difference in gene expression between the two microcarriers for the positive MSC surface markers CD-90, CD-105, and CD-73 as well as showing either low or no signal for negative MSC surface markers CD-45 and CD-34. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 122-133, 2019.

Dual Suppression Effect of Magnetic Induction Heating and Microencapsulation on Ice Crystallization Enables Low-Cryoprotectant Vitrification of Stem Cell-Alginate Hydrogel Constructs.

Stem cells microencapsulated in hydrogel as stem cell-hydrogel constructs have wide applications in the burgeoning cell-based medicine. Due to their short shelf life at ambient temperature, long-term storage or banking of the constructs is essential to the "off-the-shelf" ready availability needed for their widespread applications. As a high-efficiency, easy-to-operate, low-toxicity, and low-cost method for long-term storage of the constructs, low-cryoprotectant (CPA) vitrification has attracted tremendous attention recently. However, we found many cells in the stem cell-alginate constructs (∼500 μm in diameter) could not attach to the substrate post low-CPA vitrificationwith ∼2 M penetrating CPAs. To address this problem, we introduced nanowarming via magnetic induction heating (MIH) of Fe3O4 nanoparticles to minimize recrystallization and devitrification during the warming step of the low-CPA vitrification procedure. Our results indicate that high-quality stem cell-alginate hydrogel constructs with an intact microstructure, high immediate cell survival (>80%), and greatly improved attachment efficiency (by nearly three times, 68% versus 24%) of the encapsulated cells could be obtained post-cryopreservation with nanowarming. Moreover, the cells encapsulated in the cell-hydrogel constructs post-cryopreservation maintained normal proliferation under 3D culture and retained intact biological function of multilineage differentiation. This novel low-CPA vitrification approach for cell cryopreservation enabled by the combined use of alginate hydrogel microencapsulation and Fe3O4 nanoparticles-mediated nanowarming may be valuable in facilitating the widespread application of stem cells in the clinic.

Production of Adult Human Synovial Fluid-Derived Mesenchymal Stem Cells in Stirred-Suspension Culture

The chondrogenic potential of synovial fluid-derived mesenchymal stem cells (SF-MSCs) supports their use in cartilage regeneration strategies. However, their paucity in synovial fluid necessitates their proliferation in culture to generate clinically relevant quantities. Here it was determined that 125 mL stirred suspension bioreactors utilizing Cytodex-3 microcarrier beads represent a viable platform for the proliferation of these cells. During the inoculation phase, a bead loading of 2 g/L, an inoculation ratio of 4.5 cells/bead, and continuous agitation at 40 rpm in a medium with 5% serum resulted in high cell attachment efficiencies and a subsequent overall cell fold expansion of 5.7 over 8 days. During the subsequent growth phase, periodic addition of new microcarriers and fresh medium increased culture longevity, resulting in a 21.3 cell fold increase over 18 days in the same vessel without compromising the defining characteristics of the cells. Compared to static tissue culture flasks, a bioreactor-based bioprocess requires fewer handling steps, is more readily scalable, and for the same cell production level, has a lower operating cost as it uses approximately half the medium. Therefore, stirred suspension bioreactors incorporating microcarrier technology represent a viable and more efficient platform than tissue culture flasks for the generation of SF-MSCs in culture.

Kinetic analysis of cell decay during the filling process: Application to lot size determination in manufacturing systems for human induced pluripotent and mesenchymal stem cells

Scale-up of a cell-manufacturing system involves an extension of the filling process time because of the increased number of vials to be filled with the cells suspended in a cryopreservation solution. As the solution includes cryoprotective agents such as dimethyl sulfoxide that is toxic to cells, a longer processing time has an adverse impact on the quality of cell products. Therefore, understanding the related cell-decay kinetics is a major challenge for the development of robust and scalable cell-manufacturing systems. In this study, a new set of performance indices were defined to quantify the state and potential of cells suspended in a cryopreservation solution, i.e., the survival ratio of cells suspended in cryopreservation solution was provided for the cell state estimation, and the recovery ratio of cells after freezing and thawing, the attachment efficiency of cells after seeding, and the specific growth rate of surviving cells were provided for the cell potential estimation. Using these indices, the time-dependencies of cell viability variation in human induced pluripotent (hiPSCs) and human mesenchymal stem cells (hMSCs) suspended in a cryopreservation solution were investigated. Based on the obtained kinetic data, strategies for developing and controlling cell-manufacturing systems were discussed.

Effect of magnesium on the osteogenesis of normal human osteoblasts.

Biomaterials containing magnesium are used for implants and bone regeneration. However, mechanisms underlying the biologic effects of magnesium are still largely unknown and have not been examined on normal human osteoblasts. This study was designed to test the effect of supplemented Mg2+ concentrations between 0.5mM and 16mM on the osteogenic behaviors of normal human primary osteoblasts. Human primary osteoblasts were cultured in the groups with various concentrations of supplemented magnesium for various time intervals. Cell proliferation was measured using crystal violet staining. Degree of Alkaline Phosphatase (ALP) activity was measured by fluorometric assay. Expression of osteocalcin was measured by immunosorbent assay. Mineralization of cultures was determined by Alizarin Red S staining. Results showed that initial cell attachment efficiency was not affected by supplemented Mg2+ (P>0.05). At 21 days, proliferation rates increased in groups containing 0.5mM-4mM supplemented Mg2+ and decreased in groups of supplemented 8mM and 16mM Mg2+. ALP activity and osteocalcin expression were upregulated in groups of supplemented Mg2+ between 0.5mM-2.0mM (P<0.05), but downregulated in groups with supplemented Mg2+ concentrations of 4mM and above (P<0.05). Cultures with 1mM and 2mM supplemented Mg2+ showed upregulated mineralization activity compared to the control (P<0.05), but downregulated in groups with supplemented Mg2+ concentrations of 4mM and above (P<0.05). The present study based on an experimental design demonstrated the impact of 2mM supplemented Mg2+ on induced-proliferation and differentiation of normal human osteoblasts.