Prolonged Culture Period Research Articles

Safety and clinical efficacy of rapidly-generated trivirus-directed T cells as treatment for adenovirus, EBV, and CMV infections after allogeneic hematopoietic stem cell transplant

Adoptive transfer of virus-specific T cells can prevent and treat serious infections with Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus (Adv) after allogeneic hematopoietic stem cell transplant. It has, however, proved difficult to make this approach widely available since infectious virus and viral vectors are required for T cell activation, followed by an intensive and prolonged culture period extending over several months. We now show that T cells targeting a range of viral antigens derived from EBV, CMV, and Adv can be reproducibly generated in a single culture over a 2-3-week period, using methods that exclude all viral components and employ a much-simplified culture technology. When administered to recipients of haploidentical (n = 5), matched unrelated (n = 3), mismatched unrelated (n = 1) or matched related (n = 1) transplants with active CMV (n = 3), Adv (n = 1), EBV (n = 2), EBV+Adv (n = 2) or CMV+Adv (n = 2) infections, the cells produced complete virological responses in 80%, including all patients with dual infections. In each case, a decrease in viral load correlated with an increase in the frequency of T cells directed against the infecting virus(es); both immediate and delayed toxicities were absent. This approach should increase both the feasibility and applicability of T cell therapy. The trial was registered at www.clinicaltrials.gov as NCT01070797.

Long-term culture of primary porcine oviduct epithelial cells: Validation of a comprehensive in vitro model for reproductive science

Recently, we established a protocol for the cultivation of primary porcine oviduct epithelial cells (POEC), which promoted tissue-like morphology for a prolonged culture period. The present study focuses on developing this model into a comprehensive, standardized culture system, as a candidate tool for reproductive toxicity testing and basic research. We cultivated POEC isolated from 25 animals in our culture system for both 3 and 6 weeks and systematically analyzed effects of medium conditioning, supplementation with standardized sera, and culture duration in both freshly isolated and cryopreserved cells. The differentiation status was evaluated via histomorphometry, transepithelial electrical resistance (TEER) measurement, and expression analyses. The culture system possessed high reproducibility, more than 95% of cultures achieved a fully differentiated phenotype. Cells recapitulated in vivo–like morphology and ultrastructure from 3 to 6 weeks. Cryopreservation of the cells prior to cultivation did not affect culture quality of POEC. Employment of conditioned medium ensured optimal promotion of POEC differentiation, and different standardized sera induced fully differentiated phenotypes. Consistent TEER establishment indicated the presence and maintenance of cell type–specific intercellular junctions. The functionality of POEC was proven by consistent mucin secretion and stable expression of selected markers over the whole culture duration. We conclude that POEC are suitable for experiments from 3 weeks up to at least 6 weeks of culture. Therefore, this culture system could be used for in vitro estrous cycle simulation and long-term investigation of toxic effects on oviduct epithelium.

Restoration of keratocyte cell phenotype through epithelial‐stromal cell interactions in a 3D hydrogel model

AbstractPurpose In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. In vitro co‐culturing aims to recapture this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. The aim of this study was to investigate epithelial‐stromal cell signalling for the control of corneal stromal cell plasticity in 3D culture models.Methods Corneal stromal cells were activated to their injury phenotype (fibroblasts) before being encapsulated in 3D collagen hydrogels. 3 different epithelial‐stromal co‐culture methods were then examined; epithelial explant; transwell; the use of conditioned media. Using non‐destructive monitoring tools we revealed how epithelial co‐culturing affects stromal cell differentiation in terms of construct contraction and elastic modulus measurements in a 3D collagen hydrogel environment for prolonged culture periods. Cell viability, phenotype, morphology and protein expression was investigated to corroborate our mechanical findings.Results It was shown that activated stromal cells retain their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a non‐activated keratocyte cell lineage in terms of behaviour and biological properties; and then back again via TGF‐β1 media supplementation. It was then revealed that epithelial‐stromal interactions can be blocked via the use of wortmannin inhibition.Conclusion A greater understanding of stromal‐epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury whereby such interactions are vital.

Influence of in vitro biomimicked stem cell ‘niche’ for regulation of proliferation and differentiation of human bone marrow-derived mesenchymal stem cells to myocardial phenotypes: serum starvation without aid of chemical agents and prevention of spontan

Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow-derived mesenchymal stem cells (hBM-MSCs) undergo senescence-related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5-azacytidine was not associated with the phenomenon, but the serum-starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM-MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle-shape morphology, increased in proliferation rate by two-fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques.

EBV-transformed lymphoblastoid cell lines as vaccines against cancer testis antigen-positive tumors.

EBV-transformed lymphoblastoid cell lines (LCL) are potent antigen-presenting cells. To investigate their potential use as cancer testis antigen (CTA) vaccines, we studied the expression of 12 cancer testis (CT) genes in 20 LCL by RT-PCR. The most frequently expressed CT genes were SSX4 (50 %), followed by GAGE (45 %), SSX1 (40 %), MAGE-A3 and SSX2 (25 %), SCP1, HOM-TES-85, MAGE-C1, and MAGE-C2 (15 %). NY-ESO-1 and MAGE-A4 were found in 1/20 LCL and BORIS was not detected at all. Fifteen of 20 LCL expressed at least one antigen, 9 LCL expressed ≥2 CT genes, and 7 of the 20 LCL expressed ≥4 CT genes. The expression of CT genes did not correlate with the length of in vitro culture, telomerase activity, aneuploidy, or proliferation state. While spontaneous expression of CT genes determined by real-time PCR and Western blot was rather weak in most LCL, treatment with DNA methyltransferase 1 inhibitor alone or in combination with histone deacetylase inhibitors increased CTA expression considerably thus enabling LCL to induce CTA-specific T cell responses. The stability of the CT gene expression over prolonged culture periods makes LCL attractive candidates for CT vaccines both in hematological neoplasias and solid tumors.

In vitro differentiation of near‐unlimited numbers of functional mouse basophils using conditional Hoxb8

Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. A method is described to massively expand bone marrow-derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.

Heart repair by cardiac reprogramming

Heart repair by cardiac reprogramming

Studies on the Anti-Tumor Action of Chelate-Setting Apatite Cements

Chelate-setting apatite cement is a novel biomaterial developed as a bone substitute. We previously reported a chelate-setting apatite cement, IP6-HAp, which exhibits anti-tumor activity via apoptotic cell death. However, our preliminary data showed that excess IP6 arrests osteoblast growth. We found that a high, transient amount of IP6 was released from the cement. We therefore hypothesized that a high performance cement specific for tumor cells can be developed by controlling the release of IP6 from the cement. To validate this, we used a murine calvarial osteoblast cell line (MC3T3-E1) and a human osteosarcoma cell line (HOS). Culturing HOS or MC3T3-E1 in medium containing various concentrations of IP6 more effectively arrested the growth of HOS than that of MC3T3-E1. Although the proliferation of osteoblasts was suppressed at early growth stages in response to the release of IP6 from the cements, there was no difference in the number of cells after a prolonged culture period. In contrast, osteosarcoma cell growth remained suppressed even after a prolonged culture period. To better understand why these two cell types respond differently to IP6, we investigated cell viability by measuring the ratio of living and dead cells. Our findings suggest that this novel bone graft cement will find unique uses due the different sensitivity of tumor cells and osteoblasts towards IP6.

The Potential of Prolonged Tissue Culture to Reduce Stress Generation and Retraction in Engineered Heart Valve Tissues

In tissue-engineered (TE) heart valves, cell-mediated processes cause tissue compaction during culture and leaflet retraction at time of implantation. We have quantified and correlated stress generation, compaction, retraction, and tissue quality during a prolonged culture period of 8 weeks. Polyglycolic acid/poly-4-hydroxybutyrate strips were seeded with vascular-derived cells and cultured for 4-8 weeks. Compaction in width, generated force, and stress was measured during culture. Retraction in length, generated force, and stress was measured after release of constraints at weeks 4, 6, and 8. Further, the amount of DNA, glycosaminoglycans (GAGs), collagen, and collagen cross-links was assessed. During culture, compaction and force generation increased to, respectively, 63.9% ± 0.8% and 43.7 ± 4.3 mN at week 4, after which they remained stable. Stress generation reached 27.7 ± 3.2 kPa at week 4, after which it decreased to ∼8.5 kPa. At release of constraints, tissue retraction was 44.0% ± 3.7% at week 4 and decreased to 29.2% ± 2.8% and 26.1% ± 2.2% at, respectively, 6 and 8 weeks. Generated force (8-16 mN) was lower at week 6 than at weeks 4 and 8. Generated stress decreased from 11.8 ± 0.9 kPa at week 4 to 1.4 ± 0.3 and 2.4 ± 0.4 kPa at, respectively, weeks 6 and 8. The amount of GAGs increased at weeks 6 and 8 compared to week 4 and correlated to the reduced stress and retraction. In summary, prolonged culture resulted in decreased stress generation and retraction, likely as a result of the increased amount of GAGs. These results demonstrate the potential of prolonged tissue culture in developing functional, nonretracting, TE heart valves.

Adult Human Brain Neural Progenitor Cells (NPCs) and Fibroblast-Like Cells Have Similar Properties In Vitro but Only NPCs Differentiate into Neurons

The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC) culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP) of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia), and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs). These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5–6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments.

Arterial shear stress augments the differentiation of endothelial progenitor cells adhered to VEGF-bound surfaces

Our ongoing studies show that vascular endothelial cell growth factor (VEGF)-bound surfaces selectively capture endothelial progenitor cells (EPCs) in vitro and in vivo, and that surface-bound VEGF stimulates intracellular signal transduction pathways over prolonged culture periods, resulting in inductive differentiation of EPCs. In this article, we investigated whether simulated arterial shear stress augments the differentiation of EPCs adhered to a VEGF-bound surface. Human peripheral blood-derived mononuclear cells adhered to a VEGF-bound surface were exposed to 1 day of shear stress (15dynes/cm2, corresponding to shear load in arteries). Shear stress suppressed the expression of mRNAs encoding CD34 and CD133, which are markers for EPCs, and augmented the expression of mRNAs encoding CD31 and von Willebrand factor (vWF) as well as vWF protein, which are markers for endothelial cells (ECs). Shear stress enhanced expression of ephrinB2 mRNA, a marker for arterial ECs, but did not significantly change expression of EphB4 mRNA, a marker for venous ECs. Focused protein array analysis showed that mechanotransduction by shear stress activated the p38 and MAPK pathways in EPCs. Thus, arterial shear stress, in concert with surface-bound VEGF, augments the differentiation of EPCs. These results strongly support previous observation of rapid differentiation of EPCs captured on VEGF-bound stents in a porcine model.

Bone cells from patients with quiescent Crohn's disease show a reduced growth potential and an impeded maturation

Patients with Crohn's disease (CD) are at increased risk of developing osteoporosis. The mechanism underlying bone loss in CD patients is only partly understood. Inflammation is thought to contribute by causing a disturbed bone remodeling. In this study, we aimed to compare functional characteristics of osteoblasts from CD patients and controls, as osteoblasts are one of the effector cells in bone remodeling. The study included 18 patients with quiescent CD and 18 healthy controls. Bone cells obtained from iliac crest biopsies were cultured in the absence and presence of the inflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α, IL-10, and TGF-β. At various time points, cell proliferation and differentiation were analyzed. Bone cells from CD patients showed a prolonged culture period to reach confluence and a decreased cell number at confluence. CD patient-derived bone cell cultures produced higher alkaline phosphatase levels, whereas osteocalcin levels were considerably reduced compared to control cultures. At the proliferation level, the responsiveness to inflammatory cytokines was similar in bone cells from CD patients and controls. At the differentiation level, CD cultures showed an increased responsiveness to IL-6 and a decreased responsiveness to TGF-β. Responsiveness to the other cytokines tested was unaffected. In summary, we show a reduced growth potential and impeded maturation of bone cells from quiescent CD patients in vitro. These disease-related alterations combined with an unchanged sensitivity of CD patient-derived bone cells to inflammatory cytokines, provide a new insight in the understanding of CD-associated bone loss.

Epiphyseal Chondroprogenitors Provide a Stable Cell Source for Cartilage Cell Therapy.

Articular cartilage regeneration poses particularly tough challenges for implementing cell-based therapies. Many cell types have been investigated looking for a balanced combination of responsiveness and stability, yet techniques are still far from defining a gold standard. The work presented focuses on the reliable expansion and characterization of a clinical grade human epiphyseal chondroprogenitor (ECP) cell bank from a single tissue donation. A parental human ECP cell bank was established, which provides the seed material for master and working cell banks. ECPs were investigated at both low and high cumulative population doublings looking at morphology, monolayer expansion kinetics, resistance to cryogenic shock, colony-forming efficiency, and cell surface markers. Three-dimensional micropellet assays were used to determine spontaneous extracellular matrix deposition at varying population doublings and monolayer 2D differentiation studies were undertaken to assess the propensity for commitment into other lineages and their stability. ECPs exhibited remarkable homogeneity in expansion with a steady proliferative potential averaging three population doublings over 8 days. Surface marker analysis revealed no detectable contaminating subpopulations or population enrichment during prolonged culture periods. Despite a slight reduction in Sox9 expression levels at higher population doublings in monolayer, nuclear localization was equivalent both in monolayer and in micropellet format. Equally, ECPs were capable of depositing glycosaminoglycans and producing aggrecan, collagen I, and collagen II in 3D pellets both at low and high population doublings indicating a stable spontaneous chondrogenic potential. Osteogenic induction was differentially restricted in low and high population doublings as observed by Von Kossa staining of calcified matrix, with a notable collagen X, MMP13, and ADAMTS5 downregulation. Rare adipogenic induction was seen as evidenced by cytoplasmic lipid accumulation detectable by Oil Red O staining. These findings highlight the reliability, stability, and responsiveness of ECPs over prolonged culture, making them ideal candidates in defining novel strategies for cartilage regeneration.

Tissue Engineering for the Neonatal and Pediatric Patients

Of all the surgical specialties, the remit of the pediatric surgeon encompasses the widest range of organ systems and includes disorders from the fetus to the adolescent. As such, the recent emergence of tissue engineering is of particular interest to the pediatric surgical community. The individual challenges of tissue engineering depend largely on the nature and function of the target tissue. In general, the main issues currently under investigation include the sourcing of an appropriate cell source, design of biomaterials for guided tissue growth, provision of a biomolecular stimulus to enhance cellular functions and the development of bioreactors to allow for prolonged periods of cell culture under specific physiological conditions. This review aims to provide a general overview of tissue engineering in the major organ systems, including the cardiovascular, digestive, urinary, respiratory, musculoskeletal, nervous, integumentary and lymphatic systems. Special attention is paid to pediatrics as well as recent clinical applications.

메기(Silurus asotus)에 투여한 멜라민의 체내함량 변화

In 2007, pet food contaminated with melamine caused hundreds of dogs and cats to develop renal failure all over the world. In 2008, over 294,000 infants consumed infant formula and developed kidney stones in China. Further investigation revealed that fish feed also contained melamine; this raised concerns about melamine residues in edible fish tissues, which could have caused the urinary tract stone epidemic. In Korea, catfish fed with assorted feed that included cuttlefish organs that contained melamine developed whitening syndrome and fell dead in some populations in 2008. This event raised suspicions about the toxicity of melamine and all feeds containing melamine were immedi-ately recalled. In this study, we investigated the rates of melamine accumulation and depletion in muscle and viscera of catfish to propose proper withdrawal periods. One group of catfish was fed a commercially available diet that con-tained 30, 100 and 300 mg melamine per kg diet for 14 days. To investigate residual melamine contents in muscle and viscera, other experimental groups were fed a melamine free diet after being fed melamine for 7 days. The residual amount of melamine was analyzed by LC-MS/MS. The melamine concentration in muscle was estimated to be 3.7 mg/kg after 6 days of feeding with a diet containing 300 mg melamine/kg. After 2 days of culture with a melamine free diet, the residual melamine was depleted and the concentration had decreased from 1.15 mg/kg to 0.19 mg/kg in the muscle of catfish fed a diet containing 300 mg melamine/kg for 7 days. The residual amount of melamine was reduced to 0.03 mg/kg in muscle after 7 days of culture with a melamine free diet and was undetectable after a pro-longed culture period of 14 days. Catfish tend to excrete melamine rapidly after oral administration and changes in body color were not observed during the short dosing period. Key words: Melamine, Catfish, LC-MS/MS

Encapsulation and Survival of a Chondrocyte Cell Line within Xanthan Gum Derivative

A chemical derivative of xanthan gum polysaccharide is investigated as a new artificial matrix for the encapsulation of chondrocytic cells. Toward this goal, a novel micro-droplet generator is developed to produce microcapsules. Microcapsules with an average diameter of 500 µm, smooth surface, and homogeneous size distribution are obtained. ATDC5 cells encapsulated in carboxymethyl xanthan (CMX) microcapsules remain viable and are observed to proliferate for prolonged culture periods with enhanced metabolic activity. Furthermore, retention of the chondrogenic phenotype is exhibited by the cells within CMX, suggesting the ability of this material to be applied in cell-delivery therapies.

Noninvasive Evaluation of Contractile Behavior of Cardiomyocyte Monolayers Based on Motion Vector Analysis

A noninvasive method for the characterization of cardiomyocyte contractile behavior is presented. Light microscopic video images of cardiomyocytes were captured with a high-speed camera, and motion vectors (which have a velocity dimension) were calculated with a high spatiotemporal resolution using a block-matching algorithm. This method could extract contraction and relaxation motions of cardiomyocytes separately and evaluate characteristics such as the beating rate, orientation of contraction, beating cooperativity/homogeneity in the monolayer, and wave propagation of impulses. Simultaneous phase-contrast imaging and calcium (Ca2+) fluorescence measurements confirmed that the timing of the maximum shortening velocity of cardiomyocytes correlated well with intracellular Ca2+ transients. Based on our analysis, gap junction inhibitors, 1-heptanol (2 mM) or 18-β-glycyrrhetinic acid (30 μM), resulted in clear changes in beating cooperativity and the propagation pattern of impulses in the cardiomyocyte monolayer. Additionally, the time dependence of the motion vector length indicated a prolonged relaxation process in the presence of potassium (K+) channel blockers, dl-sotalol (1 μM), E-4031 (100 nM), or terfenadine (100 nM), reflecting the prolonged QT (Q wave and T wave) interval of cardiomyocytes. Effects of autonomic agents (acetylcholine or epinephrine [EPI]) or EPI and propranolol on cardiomyocytes were clearly detected by the alterations of beating rate and the motion vector length in contraction and relaxation processes. This method was noninvasive and could sensitively evaluate the contractile behavior of cardiomyocytes; therefore, it may be used to study and/or monitor cardiomyocyte tissue during prolonged culture periods and in screens for drugs that may alter the contraction of cardiomyocytes.

Dual effects of heparin on BMP-2-induced osteogenic activity in MC3T3-E1 cells

AbstractHeparin displays several types of biological activities by binding to various extracellular molecules, including pivotal roles in bone metabolism. We have previously reported that heparin competitively inhibits the binding activity of bone morphogenic protein-2 (BMP-2) to BMP and the BMP receptor (BMPR) and suppresses BMP-2 osteogenic activity. In the present study, we examined whether heparin affects osteoblast differentiation induced by BMP-2 at various time points in vitro. We found that 72h of treatment with heparin inhibited alkaline phosphatase (ALP) activity. However, 144h of treatment enhanced the ALP activity in BMP-2-stimulated MC3T3-E1 cells. Although heparin decreased the phosphorylation of Smad1/5/8 after 0.5h of culture, prolonged periods of culture with heparin enhanced the Smad phosphorylation. In addition, 72h of treatment with heparin enhanced the mRNA expression of runx2 and osterix in BMP-2-stimulated MC3T3-E1 cells. Furthermore, the mRNA expression of BMP antagonists and inhibitory Smads induced by BMP-2 was preferentially blocked by heparin at the 24 and 48h time points. These findings indicate biphasic effects of heparin on BMP-2 activity and suggest that heparin has complex effects on the BMP-2 osteogenic bioactivities. Prolonged culture with heparin stimulated BMP-2-induced osteogenic activity via down-regulation of BMP-2 antagonists and inhibitory Smads.

Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

Influence of cell and collagen concentration on the cell–matrix mechanical relationship in a corneal stroma wound healing model

The effect of different collagen and cell concentrations on the mechanical and remodeling behaviors of corneal stroma wound healing models consisting of collagen hydrogels seeded with human corneal fibroblasts during a 25 day culture period were examined. Human corneal fibroblasts were seeded at 1 × 10 5, 3 × 10 5 or 5 × 10 5 cells per hydrogel, and collagen concentrations of 2.5 mg/ml, 3.5 mg/ml or 4.5 mg/ml were examined. Two non-destructive techniques, spherical indentation and optical coherence tomography, were used to measure the elastic modulus and dimensional changes respectively at several time-points over the culture period. The elastic modulus of the hydrogels increased continuously over 25 days. Hydrogels with higher initial cell seeding densities and lower initial collagen concentrations were found to increase in elastic modulus faster and possessed a higher elastic modulus by the end of the culture period when compared to the other hydrogels. A mathematical equation was applied to accurately fit the change in elastic modulus over time. This study demonstrates a robust in vitro technique able to monitor the effect of different parameters on the cell–matrix mechanical relationship in a corneal stroma model during prolonged culture periods and enhances our understanding on corneal wound healing processes.