Suspension Culture Systems Research Articles

Adeno-associated virus vector-based gene therapy for hereditary diseases: current problems of application and approaches to solve them

INTRODICTION. Currently, gene therapy based on adeno-associated virus (AAV) vectors faces a number of barriers, both biomedical and technological, which require studying and overcoming for further development of this gene therapy technology.AIM. This study aimed to analyse the use of gene therapy for a range of hereditary diseases, taking into account the barriers associated with its side effects and insufficient efficacy, the determination of the therapeutic window, and individual characteristics relevant to a particular hereditary disease; additionally, the study aimed to review the approaches to lifting these barriers and increasing the availability of gene therapy through the improvement of technological approaches to production and the reduction of production costs.DISCUSSION. The authors reviewed the experience accumulated for gene therapy products that were approved or undergoing clinical trials. The study included a gene therapy applicability assessment using several hereditary diseases as a case study. The assessment showed that correct determination of the therapeutic window for a medicinal product and timely diagnosis of a hereditary disease were essential for effective and safe gene therapy. The study considered the strategies used to reduce the risks of adverse events and increase the effectiveness of AAVbased gene therapy. The authors assessed technological advancements in the manufacturing of AAV-based gene therapy products. The most perspective directions were the transition to suspension culture systems, the improvement of bioreactors, the use of new methods and materials for the purification of viral particles, the improvement of transfection systems, and the creation of new host cell lines. Ultimately, this can lead to lower production costs and an increased availability of gene therapy.CONCLUSION. Currently, gene therapy is used only for a small range of hereditary diseases. Significant barriers to its use are due to insufficient efficacy, risks of adverse events, and high costs for treatment. Ongoing biomedical and technological development should lift many of these barriers and increase access to gene therapy.

Differentiation of stem cell-derived pancreatic progenitors into insulin-secreting islet clusters in a multiwell-based static 3D culture system

Orbital shaker-based suspension culture systems have been in widespread use for differentiating human pluripotent stem cell (hPSC)-derived pancreatic progenitors toward islet-like clusters during endocrine induction stages. However, reproducibility between experiments is hampered by variable degrees of cell loss in shaking cultures, which contributes to variable differentiation efficiencies. Here, we describe a 96-well-based static suspension culture method for differentiation of pancreatic progenitors into hPSC-islets. Compared with shaking culture, this static 3D culture system induces similar islet gene expression profiles during differentiation processes but significantly reduces cell loss and improves cell viability of endocrine clusters. This static culture method results in more reproducible and efficient generation of glucose-responsive, insulin-secreting hPSC-islets. The successful differentiation and well-to-well consistency in 96-well plates also provides a proof of principle that the static 3D culture system can serve as a platform for small-scale compound screening experiments as well as facilitating further protocol development.

Agarose/crystalline nanocellulose (CNC) composites promote bone marrow-derived mast cell integrity, degranulation and receptor expression but inhibit production of de novo synthesized mediators.

Introduction: Mast cells are highly granulated tissue-resident leukocytes that require a three-dimensional matrix to differentiate and mediate immune responses. However, almost all cultured mast cells rely on two-dimensional suspension or adherent cell culture systems, which do not adequately reflect the complex structure that these cells require for optimal function. Methods: Crystalline nanocellulose (CNC), consisting of rod-like crystals 4-15nm in diameter and 0.2-1µm in length, were dispersed in an agarose matrix (12.5% w/v), and bone marrow derived mouse mast cells (BMMC) were cultured on the agarose/CNC composite. BMMC were activated with the calcium ionophore A23187 or immunoglobulin E (IgE) and antigen (Ag) to crosslink high affinity IgE receptors (FcεRI). Results: BMMC cultured on a CNC/agarose matrix remained viable and metabolically active as measured by reduction of sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT), and the cells maintained their membrane integrity as analyzed by measuring the release of lactate dehydrogenase (LDH) and propidium iodide exclusion by flow cytometry. Culture on CNC/agarose matrix had no effect on BMMC degranulation in response to IgE/Ag or A23187. However, culture of BMMC on a CNC/agarose matrix inhibited A23187-and IgE/Ag-activated production of tumor necrosis factor (TNF) and other mediators such as IL-1β, IL-4, IL-6, IL-13, MCP-1/CCL2, MMP-9 and RANTES by as much as 95%. RNAseq analysis indicated that BMMC expressed a unique and balanced transcriptome when cultured on CNC/agarose. Discussion: These data demonstrate that culture of BMMCs on a CNC/agarose matrix promotes cell integrity, maintains expression of surface biomarkers such as FcεRI and KIT and preserves the ability of BMMC to release pre-stored mediators in response to IgE/Ag and A23187. However, culture of BMMC on CNC/agarose matrix inhibits BMMC production of de novo synthesized mediators, suggesting that CNC may be altering specific phenotypic characteristics of these cells that are associated with late phase inflammatory responses.

Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier.

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries.

Dextran sulfate prevents excess aggregation of human pluripotent stem cells in 3D culture by inhibiting ICAM1 expression coupled with down-regulating E-cadherin through activating the Wnt signaling pathway

BackgroundHuman pluripotent stem cells (hPSCs) have great potential in applications for regenerative medicine and drug development. However, 3D suspension culture systems for clinical-grade hPSC large-scale production have been a major challenge. Accumulating evidence has demonstrated that the addition of dextran sulfate (DS) could prevent excessive adhesion of hPSCs from forming larger aggregates in 3D suspension culture. However, the signaling and molecular mechanisms underlying this phenomenon remain elusive.MethodsBy using a cell aggregate culture assay and separating big and small aggregates in suspension culture systems, the potential mechanism and downstream target genes of DS were investigated by mRNA sequence analysis, qRT-PCR validation, colony formation assay, and interference assay.ResultsSince cellular adhesion molecules (CAMs) play important roles in hPSC adhesion and aggregation, we assumed that DS might prevent excess adhesion through affecting the expression of CAMs in hPSCs. As expected, after DS treatment, we found that the expression of CAMs was significantly down-regulated, especially E-cadherin (E-cad) and intercellular adhesion molecule 1 (ICAM1), two highly expressed CAMs in hPSCs. The role of E-cad in the adhesion of hPSCs has been widely investigated, but the function of ICAM1 in hPSCs is hardly understood. In the present study, we demonstrated that ICAM1 exhibited the capacity to promote the adhesion in hPSCs, and this adhesion was suppressed by the treatment with DS. Furthermore, transcriptomic analysis of RNA-seq revealed that DS treatment up-regulated genes related to Wnt signaling resulting in the activation of Wnt signaling in which SLUG, TWIST, and MMP3/7 were highly expressed, and further inhibited the expression of E-cad.ConclusionOur results demonstrated that DS played an important role in controlling the size of hPSC aggregates in 3D suspension culture by inhibiting the expression of ICAM1 coupled with the down-regulation of E-cad through the activation of the Wnt signaling pathway. These results represent a significant step toward developing the expansion of hPSCs under 3D suspension condition in large-scale cultures.

Development of a 48-Well Dynamic Suspension Culture System for Pancreatic Differentiation from Human Embryonic Stem Cells.

Human pluripotent stem cells (hPSCs) have started to emerge as a potential tool with application in fields of drug discovery, disease modelling and cell therapy. A variety of protocols for culturing and differentiating pluripotent stem cells into pancreatic β like cells have been published. However, small-scale dynamic suspension culture systems, which could be applied toward systematically optimizing production strategies for cell replacement therapies to accelerate the pace of their discovery and development toward the clinic, are overlooked. Human embryonic stem cell (hESC) line H9 was used to establish the novel 48-well dynamic suspension culture system. The effects of various rotational speeds and culture medium volumes on cell morphology, cell proliferation, cell viability and cell phenotype were evaluated. Effect of cell density on the pancreatic differentiation efficiency from H9 cells in 48-well plates was further investigated. In vitro the function of pancreatic β like cells was assessed by measuring glucose-stimulated insulin secretion. A 48-well dynamic suspension culture system for hESC expansion as cell aggregates was developed. With optimized rotational speed and culture medium volume, hESCs maintained normal karyotype, viability and pluripotency. Furthermore, the system can also support the hESC aggregates subsequent differentiation into functional pancreatic β like cells after optimizing initial cell seeding density. A controllable 48-well suspension culture system in microplates for hESCs maintenance, expansion and pancreatic differentiation was developed, which may provide an efficient platform for high-throughput drug screening.

Enabling development of commercial-ready lentiviral vector manufacturing processes using stable producer cell lines

The manufacture of lentiviral vectors (LVVs) is currently a bottleneck for the development of cell and gene therapies. Typical upstream and downstream processes are often low-yielding, unscalable, involve many manual operations, and are poorly characterized. In this article, we describe the potential that LVV stable producer cell lines offer when used in upstream processes based upon suspension culture systems, and the implications that this can have on downstream unit operations. We also describe potential areas for innovation in order to develop LVV manufacturing processes suitable for commercial supply of potentially transformative cell and gene therapy medicines.

Effect of the Carbon Source and Plant Growth Regulators (PGRs) in the Induction and Maintenance of an In Vitro Callus Culture of Taraxacum officinale (L) Weber Ex F.H. Wigg

Taraxacum officinale (L.) Weber ex F.H. Wigg, commonly known as dandelion, is a cosmopolitan and perennial weed, which has medicinal properties. In vitro propagation methods are widely used on plants that have difficulties in cultivation and, consequently, low extraction yields of active metabolites. Thus, callus culture has been considered to be useful for the accumulation of several metabolites. In this study, we aimed to establish an efficient protocol for callus induction and maintenance of T. officinale, for which explant type, carbon source, light conditions, and nine different combinations of plant growth regulators (PGRs), such as 1-naphthaleneacetic acid (NAA) (from 0.05 to 0.5 mg/L) and 6-benzylaminopurine acid (BAP) (from 0.5 to 3.0 mg/L), were evaluated. The results showed that hypocotyls and roots from sterile seedlings are the best sources for callus induction, with 100% of callogenesis at every condition tested, and more than 95% of viability and friability. Complete darkness and a medium supplemented with sucrose at 2.3% (w/v) and 0.5 mg/L of NAA and 0.5 mg/L of BAP were the best conditions for callus induction, showing callus with low organogenesis and high friability. This study provides a basis for future studies on improving large-scale callus propagation and further establishment of suspension culture systems for commercial purposes.

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

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

Toward a Microencapsulated 3D hiPSC-Derived in vitro Cardiac Microtissue for Recapitulation of Human Heart Microenvironment Features.

The combination of cardiomyocytes (CM) and non-myocyte cardiac populations, such as endothelial cells (EC), and mesenchymal cells (MC), has been shown to be critical for recapitulation of the human heart tissue for in vitro cell-based modeling. However, most of the current engineered cardiac microtissues still rely on either (i) murine/human limited primary cell sources, (ii) animal-derived and undefined hydrogels/matrices with batch-to-batch variability, or (iii) culture systems with low compliance with pharmacological high-throughput screenings. In this work, we explored a culture platform based on alginate microencapsulation and suspension culture systems to develop three-dimensional (3D) human cardiac microtissues, which entails the co-culture of human induced pluripotent stem cell (hiPSC) cardiac derivatives including aggregates of hiPSC–CM and single cells of hiPSC–derived EC and MC (hiPSC–EC+MC). We demonstrate that the 3D human cardiac microtissues can be cultured for 15 days in dynamic conditions while maintaining the viability and phenotype of all cell populations. Noteworthy, we show that hiPSC–EC+MC survival was promoted by the co-culture with hiPSC–CM as compared to the control single-cell culture. Additionally, the presence of the hiPSC–EC+MC induced changes in the physical properties of the biomaterial, as observed by an increase in the elastic modulus of the cardiac microtissue when compared to the hiPSC–CM control culture. Detailed characterization of the 3D cardiac microtissues revealed that the crosstalk between hiPSC–CM, hiPSC–EC+MC, and extracellular matrix induced the maturation of hiPSC–CM. The cardiac microtissues displayed functional calcium signaling and respond to known cardiotoxins in a dose-dependent manner. This study is a step forward on the development of novel 3D cardiac microtissues that recapitulate features of the human cardiac microenvironment and is compliant with the larger numbers needed in preclinical research for toxicity assessment and disease modeling.

Adaptive defence-related changes in the metabolome of Sorghum bicolor cells in response to lipopolysaccharides of the pathogen Burkholderia andropogonis

Plant cell suspension culture systems are valuable for the study of complex biological systems such as inducible defence responses and aspects of plant innate immunity. Perturbations to the cellular metabolome can be investigated using metabolomic approaches in order to reveal the underlying metabolic mechanism of cellular responses. Lipopolysaccharides from the sorghum pathogen, Burkholderia andropogonis (LPSB.a.), were purified, chemically characterised and structurally elucidated. The lipid A moiety consists of tetra- and penta-acylated 1,4’-bis-phosphorylated disaccharide backbone decorated by aminoarabinose residues, while the O-polysaccharide chain consists of linear trisaccharide repeating units of [→2)-α-Rha3CMe-(1 → 3)-α-Rha-(1 → 3)-α-Rha-(1 → ]. The effect of LPSB.a. in triggering metabolic reprogramming in Sorghum bicolor cells were investigated using untargeted metabolomics with liquid chromatography coupled to mass spectrometry detection. Cells were treated with LPSB.a. and the metabolic changes monitored over a 30 h time period. Alterations in the levels of phytohormones (jasmonates, zeatins, traumatic-, azelaic- and abscisic acid), which marked the onset of defence responses and accumulation of defence-related metabolites, were observed. Phenylpropanoids and indole alkaloids as well as oxylipins that included di- and trihydroxyoctadecedienoic acids were identified as signatory biomarkers, with marked secretion into the extracellular milieu. The study demonstrated that sorghum cells recognise LPSB.a. as a ‘microbe-associated molecular pattern’, perturbing normal cellular homeostasis. The molecular features of the altered metabolome were associated with phytohormone-responsive metabolomic reconfiguration of primary and secondary metabolites originating from various metabolic pathways, in support of defence and immunity.

Activation of the RhoA-YAP-β-catenin signaling axis promotes the expansion of inner ear progenitor cells in 3D culture.

Cellular mechanotransduction plays an essential role in the development and differentiation of many cell types, but if and how mechanical cues from the extracellular matrix (ECM) influence the fate determination of inner ear progenitor cells (IEPCs) remains largely unknown. In the current study, we compared the biological behavior of IEPCs in Matrigel‐based suspension and encapsulated culture systems, and we found that the mechanical cues from the ECM promote the survival and expansion of IEPCs. Furthermore, we found that the mechanical cues from the ECM induced the accumulation of Ras homolog family member A (RhoA) and caused the polymerization of actin cytoskeleton in IEPCs. These changes in turn resulted in increased Yes‐associated protein (YAP) nuclear localization and enhanced expansion of IEPCs, at least partially through upregulating the canonical Wnt signaling pathway. We therefore provide the first demonstration that the RhoA‐YAP‐β‐catenin signaling axis senses and transduces mechanical cues from the ECM and plays crucial roles in promoting the expansion of IEPCs.

Key considerations for the use of suspension culture systems for viral vector manufacturing

Key considerations for the use of suspension culture systems for viral vector manufacturing

The effect of light, phenylalanine and methyl jasmonate, alone or in combination, on growth and secondary metabolism in cell suspension cultures of Vitis vinifera

The cultivated grapevine V. vinifera is a rich source of stilbene compounds such as resveratrol, which are widely believed to provide dietary protection against the development of cardiovascular disease and some forms of cancer. Elicitation is a well-known strategy to increase commercial production of natural products in plant cell suspension culture systems. Callus tissues obtained from berry slices of V. vinifera cv. Shahani grown on an optimized medium were used to develop cell suspension cultures used to study the effects of elicitation on stilbene synthesis. The effect of two light regimes (135.1 μmol. s−1 m−2 radiation, and dark), the concentration of phenylalanine (Phe; 0, 0.1, 0.5 and 1 mM) and of methyl jasmonate elicitor (MeJA; 0 and 25 μM), alone or in combination, were tested. The results showed that cultures grown in darkness resulted in significantly higher levels of the accumulation of total stilbenes (resveratrol + piceid) compared with the high light condition. The combined treatments of dark +1 mM Phe and dark +25 μM MeJA induced the synthesis of high levels of total phenolics, total flavonoids and total stilbenes. Finally, the combined elicitation of dark +1 mM Phe + 25 μM MeJA gave the highest synergistic coefficient (1.24) and proved to be the most effective treatment for the production of total phenolics, total flavonoids, and total stilbenes with mean contents of 384.80 mg GA/g DW, 527.62 mg catechin/g DW and 188.34 μg/g DW, respectively. The results of our study suggest that the combinations of dark together with MeJA and/or Phe can be used as an efficient method for the future scale-up of V. vinifera cell cultures for the production of high value stilbene compounds in a bioreactor system.

Pros and cons of suspension culture systems in LVV upstream bioprocessing

Pros and cons of suspension culture systems in LVV upstream bioprocessing

Local supplementation with plant-derived recombinant human FGF2 protein enhances bone formation in critical-sized calvarial defects.

Numerous studies have demonstrated the advantages of plant cell suspension culture systems in producing bioactive recombinant human growth factors. This study investigated the biological activity of recombinant basic human fibroblast growth factor (rhFGF2) protein produced by a plant culture system to enhance new bone formation in a bone defect mouse model. The human FGF2 cDNA gene was cloned into a plant expression vector driven by the rice α-amylase 3D promoter. The vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), and the clone with the highest expression of rhFGF2 was selected. Maximum accumulation of rhFGF2 protein (approximately 28mg/l) was reached at 13day post-incubation. Male C57BL/6 mice underwent calvarial defect surgery and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 5μg of plant-derived rhFGF2 (p-FGF2) protein or E. coli-derived rhFGF2 (e-FGF2) protein. Similar to the effects of e-FGF2, local delivery with p-FGF2 enhanced bone healing in the damaged region to higher levels than the ACS group. Exogenous addition of p-FGF2 or e-FGF2 exhibited similar effects on proliferation, mineralization, and osteogenic marker expression in MC3T3-E1 cells. Together, the current findings support the usefulness of this plant-based expression system for the production of biologically active rhFGF2.

Growth, morphology and maturation ability of Pinus pinea embryogenic suspension cultures

Stone pine (Pinus pinea L.) is a Mediterranean forest species used for the commercial harvesting of its edible seeds, known as pine nuts. Plant regeneration by somatic embryogenesis on semi-solid medium has been achieved in stone pine, but suspension culture systems for the large-scale production of somatic embryos need to be developed. Suspension cultures were established from several embryogenic lines. Growth parameters as settled cell volume (SCV) and dry weight (DW) in suspension cultures were influenced by genotype, initial inoculum density, orbiting speed and type of carbohydrate in the culture medium. A higher linear growth (DW) up to week four was obtained using 20 mg mL−1 inoculum. The orbiting speed affected the SCV as well as the morphology of embryogenic cultures. Growth parameters decreased when orbiting speed was increased from 50 rpm to 150 rpm. Cultures at 50 rpm most commonly grew as clusters of embryonal cell masses, whereas at 150 rpm there were large numbers of single cells. Cultures grew slowly in medium with maltose instead of sucrose, but growth was not affected when plant growth regulators (PGRs) concentration was decreased. A sustainable suspension culture could be maintained by transferring 250 mg of filtered cell mass to 25 mL of liquid medium at 3-week intervals. Culture scaling-up in liquid medium resulted in a 23-fold DW increase in 6 weeks, compared with 12 fold increase on semi-solid medium. Cotyledonary embryos were obtained on semi-solid maturation medium from lines proliferated in suspension cultures. Increased somatic embryo production was obtained when proliferation was performed at 100 rpm orbiting speed in liquid medium with low PGRs concentration and sucrose instead of maltose.

Immobilisation of Delta-like 1 ligand for the scalable and controlled manufacture of hematopoietic progenitor cells in a stirred bioreactor

BackgroundUmbilical cord blood provides a source of hematopoietic stem cells for transplantation with immunological and availability advantages over conventional bone marrow sources. Limited cell numbers and slower engraftment from umbilical cord blood units has led to the clinical development of immobilised Notch ligand Delta-Like 1 to promote ex vivo expansion of a rapidly engrafting cell population. However, current immobilisation methods are not simple to scale in a controlled manner.ResultsDelta-Like 1 was immobilised onto streptavidin coated magnetic particles via a heterobifunctionalised polyethylene glycol linker molecule to provide an easily manipulated format of surface protein presentation. CD34+ enriched cord blood cells were treated with Delta-Like 1 immobilised particles, and immunophenotypic markers measured to monitor population distributions using cluster identification, characterization, and regression software. The amenability of the approach to scalability was evaluated in a micro-scale stirred tank bioreactor. Surface concentration of Delta-Like 1 was well controlled used differing stoichiometric reagent ratios. Protein immobilisation was a cost effective process and particles were efficiently removed from the final cell product. Immobilised Delta-Like 1 is functional and stimulates qualitatively similar CD34hi, CD38lo, CD90lo, CD133hi, CD135hi progenitor expansion in both static culture and scalable stirred culture platforms.ConclusionsImmobilised Delta-Like 1 in this form has the potential to improve the manufacturing efficiency and control of final ex vivo expanded cell product through compatibility with highly controlled and characterised suspension culture systems.

Recombinant human IGF-1 produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects

Transgenic plant cell suspension culture systems have been utilized extensively as convenient and efficient expression systems for the production of recombinant human growth factors. We produced insulin-like growth factor-1 using a plant suspension culture system (p-IGF-1) and explored its effect on new bone formation in calvarial defects. We also compared the bone regenerating potential of p-IGF-1 with commercial IGF-1 derived from Escherichia coli (e-IGF-1). Male C57BL/6 mice underwent calvarial defect surgery, and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 13μg of p-IGF-1 (p-IGF-1 group) or e-IGF-1 (e-IGF-1 group). The sham group did not receive any treatment with ACS or IGFs after surgery. Live μCT and histological analyses showed critical-sized bone defects in the sham group, whereas greater bone formation was observed in the p-IGF-1 and e-IGF-1 groups than the ACS group both 5 and 10weeks after surgery. Bone mineral density, bone volume, and bone surface values were also higher in the IGF groups than in the ACS group. Local delivery of p-IGF-1 or e-IGF-1 more greatly enhanced the expression of osteoblast-specific markers, but inhibited osteoclast formation, in newly formed bone compared with ACS control group. Specifically, p-IGF-1 treatment induced higher expression of alkaline phosphatase, osteocalcin, and osteopontin in the defect site than did e-IGF-1. Furthermore, treatment with p-IGF-1, but not e-IGF-1, increased mineralization of MC3T3-E1 cells, with the attendant upregulation of osteogenic marker genes. Collectively, our findings suggest the potential of p-IGF-1 in promoting the processes required for bone regeneration.

Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.