Controlled Culture System Research Articles

The impact of cellular environment on in vitro drug screening.

There are various reasons for drug failure in the developmental stage including toxicity, adverse effects and inefficacy. This is likely due to the differences in drug behavior between a simple and controlled cell culture system to that of a more complex whole organism environment. While the use of human phenotypical cells relevant to the condition may provide more accurate screening results, they are susceptible to producing false positives as cells are continuously influenced by constant chemical and physical interaction with the surrounding microenvironment. Therefore, several microenvironmental and pharmacomechanical aspects must be factored in during tissue culture drug screening.

Utilization of microalgae, Chlorella sp. UMT LF2 for bioremediation of Litopenaeus vannamei culture system and harvesting using bio-flocculant, Aspergillus niger

Microalgae are well known as autotrophic microorganisms in aquaculture wastewater treatment, however their role in the aquaculture system is rarely explored. The purpose of this study is to evaluate the influence of adding microalgae, Chlorella sp. UMT LF2, on the functionality for bioremediation and growth performance of Whiteleg shrimp, Litopenaeus vannamei in the nursery phase reared in a control culture system. Secondly, the response surface methodology (RSM) was used to investigate further on the optimal condition for the harvesting process of excess microalgae biomass using bio-flocculant from the filamentous fungus, Aspergillus niger. The experimental set-up with Chlorella sp. additions were compared to the control group without the microalgae addition based on the water quality, microalgae biomass and shrimp growth performance. Water samples were collected and underwent in-situ and ex-situ involving nine physicochemical parameters. The results showed that introducing microalgae with zero water exchange as treatment tanks reduced the NH3, NO2− and PO43− during the intermediate until the two days before the 30-day culture period's end. Microalgae also enhanced shrimp survival and growth performance significantly 89% as compared to the control, 68%. Furthermore, the culture with the addition of microalgae maintained pH stability and enhanced dissolved oxygen during daytime in the culture water. Therefore, this finding confirmed that Chlorella sp. UMT LF2 addition is promising in improving rearing environment and nursery performance for L. vannamei in culture system. However, further research is required to demonstrate the harvesting by-product from the culture system and the potential applications.

Identification of a transcriptional signature found in multiple models of ASD and related disorders.

Epigenetic regulation plays a critical role in many neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD). In particular, many such disorders are the result of mutations in genes that encode chromatin-modifying proteins. However, although these disorders share many features, it is unclear whether they also share gene expression disruptions resulting from the aberrant regulation of chromatin. We examined five chromatin modifiers that are all linked to ASD despite their different roles in regulating chromatin. Specifically, we depleted ASH1L, CHD8, CREBBP, EHMT1, and NSD1 in parallel in a highly controlled neuronal culture system. We then identified sets of shared genes, or transcriptional signatures, that are differentially expressed following loss of multiple ASD-linked chromatin modifiers. We examined the functions of genes within the transcriptional signatures and found an enrichment in many neurotransmitter transport genes and activity-dependent genes. In addition, these genes are enriched for specific chromatin features such as bivalent domains that allow for highly dynamic regulation of gene expression. The down-regulated transcriptional signature is also observed within multiple mouse models of NDDs that result in ASD, but not those only associated with intellectual disability. Finally, the down-regulated transcriptional signature can distinguish between control and idiopathic ASD patient iPSC-derived neurons as well as postmortem tissue, demonstrating that this gene set is relevant to the human disorder. This work identifies a transcriptional signature that is found within many neurodevelopmental syndromes, helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in ASD.

Sucrose addition directionally enhances bacterial community convergence and network stability of the shrimp culture system

Sucrose is an effective carbon source for creating more reliable and environmentally friendly conditions for shrimp growth by regulating bacteria in biofloc-based culture systems. However, the influence of sucrose addition on the interaction, co-occurrence networks, and assembly mechanisms of bacterial communities in biofloc-based culture systems remains largely unknown. Here, we comprehensively investigated the effects of sucrose addition on bacterial communities in three habitats (water, bioflocs, and gut). The bacterial community structures and compositions of these three habitats became more similar in groups with sucrose addition, compared with those in controls. More than 50% gut bacterial communities were mainly derived from water and biofloc communities in the sucrose addition groups, but only about 33% bacterial communities migrated from water and biofloc to the gut in the control culture system. Sucrose addition accordantly enriched core taxa belonging to the phylum Actinobacteria and the families Rhodobacteraceae and Flavobacteriaceae in water, biofloc, and gut habitats. These core taxa were important for maintaining bacterial network stability in the sucrose addition culture systems and some were identified as keystone taxa for improving shrimp growth. Furthermore, after sucrose addition, gut bacterial community assembly from water and biofloc was dominated by the heterogeneous select with the ratios of 55–91% and 67–83%, respectively, indicating that sucrose addition can directionally shape the bacterial assembly of the shrimp culture system. These results provide a basis for selectively regulating certain beneficial taxa to improve shrimp growth in culture systems.

Novel perfusion ex-vivo culture system enables physiological and pathological studies on porcine arteries

Abstract Introduction Routine cardiovascular interventions such as cardiac bypass, balloon angioplasty and stenting provoke vascular activation and remodelling often leading to rehospitalization and further interventions. Conventional in vitro models fail to account for the complex vascular environment essential for vascular tissue fitness. Purpose Our research aims to culture whole porcine arteries in using a novel cost efficient and versatile perfusion system (EasyFlow) and identify the contribution of adventitial progenitors to post-injury remodelling. Methods EasyFlow insert was 3D printed to convert a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine arteries were excised and cultured under constant pulsatile flow for up to 7 days. Injury was performed by balloon catheter at day 0. Tissues were evaluated by doppler ultrasound, immunofluorescence and confocal imaging and PCR, at different timepoints. Results The EasyFlow adaptor takes advantage of 50 ml centrifuge tubes isolating reaction space from the environment and reducing the culture volume. The adaptability of the design facilitates the incubation of vessels of different size and origin while the self-contained perfusion allows parallel cultures and minimal media consumption. EasyFlow perfusion culture of porcine arteries preserves the endothelial coverage and the smooth muscle cell organisation, as compared to static culture. Gene expression analysis and immunofluorescence indicated an increased expression of platelet-derived growth factor receptor beta (PDGFRb) and a decrease in smooth muscle actin (SMA) in the static culture, compared to the perfusion. Furthermore, static culture showed an increased cellular activation in adventitia (proliferating cell nuclear antigen expression), as compared to perfused tissues. Balloon injury followed by flow culture recapitulated the early hallmarks of vascular remodelling, including intimal denudation indicated by loss of platelet endothelial cell adhesion molecule signal, smooth muscle cell disarray shown by loss of actin stress fibre organisation and activation in the media, as supported by confocal microscope and gene expression analysis, accompanied by significant morphological changes based on ultrasound imaging. Conclusion This work lay the basis for future investigations into the pathological remodelling of blood vessels, by providing a robust and controlled culture system for the maintenance of porcine blood vessels in culture. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Surrey En-Face Staining of Arterial Lumen

Kidney Organoid and Microphysiological Kidney Chip Models to Accelerate Drug Development and Reduce Animal Testing.

Kidneys are critical for the elimination of many drugs and metabolites via the urine, filtering waste and maintaining proper fluid and electrolyte balance. Emerging technologies incorporating engineered three-dimensional (3D) in vitro cell culture models, such as organoids and microphysiological systems (MPS) culture platforms, have been developed to replicate nephron function, leading to enhanced efficacy, safety, and toxicity evaluation of new drugs and environmental exposures. Organoids are tiny, self-organized three-dimensional tissue cultures derived from stem cells that can include dozens of cell types to replicate the complexity of an organ. In contrast, MPS are highly controlled fluidic culture systems consisting of isolated cell type(s) that can be used to deconvolute mechanism and pathophysiology. Both systems, having their own unique benefits and disadvantages, have exciting applications in the field of kidney disease modeling and therapeutic discovery and toxicology. In this review, we discuss current uses of both hPSC-derived organoids and MPS as pre-clinical models for studying kidney diseases and drug induced nephrotoxicity. Examples such as the use of organoids to model autosomal dominant polycystic kidney disease, and the use of MPS to predict renal clearance and nephrotoxic concentrations of novel drugs are briefly discussed. Taken together, these novel platforms allow investigators to elaborate critical scientific questions. While much work needs to be done, utility of these 3D cell culture technologies has an optimistic outlook and the potential to accelerate drug development while reducing the use of animal testing.

Ex vivo conditioning of peripheral blood mononuclear cells of diabetic patients promotes vasculogenic wound healing.

The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum‐free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)‐QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum‐free expansion media with VEGF, Flt‐3 ligand, TPO, IL‐6, and SCF. The vascular regeneration capability of MNC‐QQ cells pre‐ or post‐QQc was evaluated with an EPC colony‐forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 104 pre‐ and post‐MNC‐QQc cells from diabetic donors were injected into a murine wound‐healing model using Balb/c nude mice. The percentage of wound closure and angio‐vasculogenesis was then assessed. This study revealed vasculogenic, anti‐inflammatory, and wound‐healing effects of MNC‐QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound‐healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient‐based vascular and regenerative therapy for patients with diabetes mellitus.

Analysis of effects of environmental fluctuations on the marine mysid Neomysis awatschensis and its development as an experimental model animal

Mysids are experimental models and are among the most important food items for animals in aquaria and that support fisheries, and even for humans, but information on their performance in controlled culture systems is still limited. We reared the marine mysid Neomysis awatschensis in a controlled laboratory system, and measured its growth, 20–hydroxyecdysone (20E) levels, molting, and survival in response to environmental fluctuations in temperature, pH, and salinity, and inferred their potential associations based on annual field sampling. The 20E levels were significantly elevated during the postnauplioid stages, and even higher levels of 20E were maintained in the adult stages than in the nauplioid stages. Values of growth parameters (i.e. total length and the lengths of the antennal scale, expod, endopod, and telson) and 20E levels were higher during a 40-day period at 25 °C than at other temperatures, with shorter intermolt intervals, although morality was also increased. Among the surviving mysids, the number of newly hatched juveniles produced was higher for females exposed to 20 °C than that in other groups. Relatively higher growth and survival rates were measured at salinities over 25 practical salinity, while lower salinities under 15 practical salinity significantly reduced growth and survival. The number of newly hatched juveniles was lower at salinities under 15 practical salinity compared to those over 20 practical salinity. Overall, low temperature and salinity reduced mysid reproduction and the maintenance of the second generation. In the case of pH variation (pH of 7.0–8.0), there were no significant effects on growth and the number of newly hatched juveniles, although the survival rate was slightly lower and the 20E level fluctuated at a pH of 7.0. We believe that these associations between growth and environmental conditions can provide crucial information for optimizing mass mysid culture for experimental and ecotoxicological usage in the laboratory.

Characteristics of Phytoplankton Succession Based on the Functional Group in the Enclosed Culture System

The present study was conducted from August to December 2016 in a cylindrical water tank with a diameter of 1 m, a height of 4 m and a capacity of 3,000 L. The field water and sediment from the Nakdong River were also sampled for the experimental culture (field water+sediment) and control culture (field water), respectively. In this study, we aimed to investigate phytoplankton succession pattern using the phytoplankton functional group in the enclosed culture system. A total of 50 species in 27 genera including Chlorophyceae (30 species), Bacillariophyceae (11 species), Cyanophyceae (7 species), and Cryptophyceae (2 species) were identified in the experimental and control culture systems. A total of 19 phytoplankton functional groups (PFGs) were identified, and these groups include B, C, D, F, G, H1, J, K, Lo, M, MP, N, P, S1, TB, W0, X1, X2 and Y. In particular, W0, J and M groups exhibited the marked succession in the experimental culture system with higher biovolumes compared to those of the control culture system, which may be related to the internal cycling of nutrients by sediment in the experimental culture system. The principal component analyses demonstrated that succession patterns in PFG were associated with the main environmental factors such as nutrients (N, P), water temperature and light intensity in two culture systems. In conclusion, the present study showed the potential applicability of the functional group for understanding the adaptation strategies and ecological traits of the phytoplankton succession in the water bodies of Korea.

Conversion of Nonproliferating Astrocytes into Neurogenic Neural Stem Cells: Control by FGF2 and Interferon-γ.

Conversion of astrocytes to neurons, via de-differentiation to neural stem cells (NSC), may be a new approach to treat neurodegenerative diseases and brain injuries. The signaling factors affecting such a cell conversion are poorly understood, and they are hard to identify in complex disease models or conventional cell cultures. To address this question, we developed a serum-free, strictly controlled culture system of pure and homogeneous "astrocytes generated from murine embryonic stem cells (ESC)." These stem cell derived astrocytes (mAGES), as well as standard primary astrocytes resumed proliferation upon addition of FGF. The signaling of FGF receptor tyrosine kinase converted GFAP-positive mAGES to nestin-positive NSC. ERK phosphorylation was necessary, but not sufficient, for cell cycle re-entry, as EGF triggered no de-differentiation. The NSC obtained by de-differentiation of mAGES were similar to those obtained directly by differentiation of ESC, as evidenced by standard phenotyping, and also by transcriptome mapping, metabolic profiling, and by differentiation to neurons or astrocytes. The de-differentiation was negatively affected by inflammatory mediators, and in particular, interferon-γ strongly impaired the formation of NSC from mAGES by a pathway involving phosphorylation of STAT1, but not the generation of nitric oxide. Thus, two antagonistic signaling pathways were identified here that affect fate conversion of astrocytes independent of genetic manipulation. The complex interplay of the respective signaling molecules that promote/inhibit astrocyte de-differentiation may explain why astrocytes do not readily form neural stem cells in most diseases. Increased knowledge of such factors may provide therapeutic opportunities to favor such conversions. Stem Cells 2016;34:2861-2874.

Reproductive performance of seahorse, Hippocampus barbouri (Jordan and Richardson 1908) in control condition

Hippocampus barbouri is one of the seahorse species found in shallow water of Malaysia. It is used as known as a global trade species in ornamental fish industry. To date, there is no documented report on seahorse aquaculture especially for H. barbouri in Malaysia even in Southeast Asia region. Seahorse aquaculture should be considered as an alternative source of seahorses to reduce the pressure on wild population. Therefore, this study was conducted to establish suitable techniques for broodstock maintenance and reproduction by focusing on culture system and feeding. Hippocampus barbouri were maintained and bred successfully in a controlled culture system. Minimum water depth required for the spawning of H. barbouri is 38 cm. Best reproductive performances was observed in broodstock fed with post-larvae shrimp. However, frozen mysid can also be used in the culture of H. barbouri. The minimal requirement of n-3 and n-6 fatty acids for the reproduction of H. barbouri was 5.13 ± 0.04 % and 14.83 ± 0.10 % respectively.

An in vitro model of hemogenic endothelium commitment and hematopoietic production.

Adult-type hematopoietic stem and progenitor cells are formed during ontogeny from a specialized subset of endothelium, termed the hemogenic endothelium, via an endothelial-to-hematopoietic transition (EHT) that occurs in the embryonic aorta and the associated arteries. Despite efforts to generate models, little is known about the mechanisms that drive endothelial cells to the hemogenic fate and about the subsequent molecular control of the EHT. Here, we have designed a stromal line-free controlled culture system utilizing the embryonic pre-somitic mesoderm to obtain large numbers of endothelial cells that subsequently commit into hemogenic endothelium before undergoing EHT. Monitoring the culture for up to 12 days using key molecular markers reveals stepwise commitment into the blood-forming system that is reminiscent of the cellular and molecular changes occurring during hematopoietic development at the level of the aorta. Long-term single-cell imaging allows tracking of the EHT of newly formed blood cells from the layer of hemogenic endothelial cells. By modifying the culture conditions, it is also possible to modulate the endothelial cell commitment or the EHT or to produce smooth muscle cells at the expense of endothelial cells, demonstrating the versatility of the cell culture system. This method will improve our understanding of the precise cellular changes associated with hemogenic endothelium commitment and EHT and, by unfolding these earliest steps of the hematopoietic program, will pave the way for future ex vivo production of blood cells.

Extracellular environment contribution to astrogliosis-lessons learned from a tissue engineered 3D model of the glial scar.

Glial scars are widely seen as a (bio)mechanical barrier to central nervous system regeneration. Due to the lack of a screening platform, which could allow in-vitro testing of several variables simultaneously, up to now no comprehensive study has addressed and clarified how different lesion microenvironment properties affect astrogliosis. Using astrocytes cultured in alginate gels and meningeal fibroblast conditioned medium, we have built a simple and reproducible 3D culture system of astrogliosis mimicking many features of the glial scar. Cells in this 3D culture model behave similarly to scar astrocytes, showing changes in gene expression (e.g., GFAP) and increased extra-cellular matrix production (chondroitin 4 sulfate and collagen), inhibiting neuronal outgrowth. This behavior being influenced by the hydrogel network properties. Astrocytic reactivity was found to be dependent on RhoA activity, and targeting RhoA using shRNA-mediated lentivirus reduced astrocytic reactivity. Further, we have shown that chemical inhibition of RhoA with ibuprofen or indirectly targeting RhoA by the induction of extracellular matrix composition modification with chondroitinase ABC, can diminish astrogliosis. Besides presenting the extracellular matrix as a key modulator of astrogliosis, this simple, controlled and reproducible 3D culture system constitutes a good scar-like system and offers great potential in future neurodegenerative mechanism studies, as well as in drug screenings envisaging the development of new therapeutic approaches to minimize the effects of the glial scar in the context of central nervous system disease.

A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro–fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm2 on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation.

A defined, controlled culture system for primary bovine chromaffin progenitors reveals novel biomarkers and modulators.

We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells.

AN IN-VITRO SCREENING METHOD TO STUDY THE ACTIVITY POTENTIAL OF BIOFERTILIZERS BASED ON TRICHODERMA AND BACILLUS SP.

Little scientific information on efficiency of different commercial biofertilizers restricts setup of further reproducible pot or field experiments and hence, provides lack of evidence of biofertilizer application in plant growth promotion and disease suppression. In the present experiment, efficiency of four commercial Trichoderma and one Bacillus biofertilizer was screened by a bio-indicator plant, cucumber (Cucumis sativus L.) under controlled laboratory conditions. Inoculation of cucumber seeds with different commercial biofertilizers significantly increased the germination rates (ca. 20–25%) and stimulated other growth parameters. In seedling establishment test, biofertilizers inoculated cucumber seedlings showed significant higher root dry weight (ca. 32 to 96%), leaf area (ca. 60 to 140%), root length (ca. 30%) and shoot dry weight (ca. 88%) in two weeks culture period compare to that of the control. Additionally, in-vitro antagonistic activity against take-all pathogen (Gaeumannomyces graminis var. tritici, Ggt) in wheat and phosphate solubilizing activity was demonstrated for Trichoderma biofertilizer. However, in-vitro solubilization of Mn was not detected. The results suggested that the potential activity of different commercial biofertilizers could be easily screened within several days with the described rapid bio-test by increasing seed germination, and improving growth and growth related parameters of cucumber grown in nutrient solution under controlled culture system.

Lymphoid Progenitor Cells from Childhood Acute Lymphoblastic Leukemia Are Functionally Deficient and Express High Levels of the Transcriptional Repressor Gfi-1

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM) has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive. The aim of the present study was to investigate the ability of lymphoid progenitors from B-cell precursor ALL BM to proliferate and undergo multilineage differentiation. By phenotype analyses, in vitro proliferation assays, and controlled culture systems, the lymphoid differentiation potentials were evaluated in BM primitive populations from B-cell precursor ALL pediatric patients. When compared to their normal counterparts, functional stem and progenitor cell contents were substantially reduced in ALL BM. Moreover, neither B nor NK or dendritic lymphoid-cell populations developed recurrently from highly purified ALL-lymphoid progenitors, and their proliferation and cell cycle status revealed limited proliferative capacity. Interestingly, a number of quiescence-associated transcription factors were elevated, including the transcriptional repressor Gfi-1, which was highly expressed in primitive CD34+ cells. Together, our findings reveal major functional defects in the primitive hematopoietic component of ALL BM. A possible contribution of high levels of Gfi-1 expression in the regulation of the stem/progenitor cell biology is suggested.

Keeping it in the family: segregation of genetic variation in Glomus irregulare

Keeping it in the family: segregation of genetic variation in <i>Glomus irregulare</i>

Development of Serum-Free Quality and Quantity Control Culture of Colony-Forming Endothelial Progenitor Cell for Vasculogenesis

Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum-free quality and quantity control culture system for colony-forming EPCs to enhance their regenerative potential. A culture with serum-free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin-6, and Flt-3 ligand was determined as optimal quality and quantity culture (QQc) in terms of the most vasculogenic colony-forming EPC expansion, evaluated by the newly established EPC colony formation assay. The QQc of umbilical cord blood-CD133(+) cells for 7 days produced a 52.9-fold increase in total cell number and 3.28-fold frequency in definitive EPC colony development, resulting in a 203.9-fold increase in estimated total definitive EPC colony number in vitro. Pre- or post-QQc cells were intramyocardially transplanted into nude rats with myocardial infarction (MI). Echocardiographic and micromanometer-tipped conductance catheter examinations 28 days post-MI revealed significant preservation of left ventricular (LV) function in rats receiving pre- or post-QQc cells compared with those receiving phosphate-buffered saline. Assessments of global LV contractility indicated a dose-dependent effect of pre- or post-QQc cells and the superior potency of post-QQc cells over pre-QQc cells. Furthermore, immunohistochemistry showed more abundant formation of both human and rat endothelial cells and cardiomyocytes in the infarcted myocardium following transplantation of post-QQc cells compared with pre-QQc cells. Our optimal serum-free quality and quantity culture may enhance the therapeutic potential of EPCs in both quantitative and qualitative aspects for cardiovascular regeneration.

Bone marrow stromal and Schwann cells from adult rats can interact synergistically to aid in peripheral nerve repair even without intercellular contact in vitro

To better understand the use of Schwann cells (SCs) and bone marrow stromal cells (BMSCs) together for nerve repair, we studied whether interactions between these two cell types by diffusible molecules can enhance their utility. In the present study, a co-culture system was established to allow BMSCs and SCs grow in the same culture medium but without physical contact. Before co-culture, the adult SCs were expanded until confluent. The adult BMSCs were cultured until P10 with CD29 and CD44 positive but CD45 negative. After 4 days in culture, > 80% of the BMSCs in the co-culture system showed both GFAP- and S-100-positive, but < 6% of the BMSCs in control culture system showed both GFAP- and S-100-positive. Meanwhile, 68.76% of the SCs in co-culture system showed S-100-positive, which was > 42.03% of the SCs in control culture system. Furthermore, the in vivo study also confirmed that differentiated BMSCs exert a more beneficial effect on repairing injured sciatic nerve function and axonal regeneration than undifferentiated BMSCs. These results indicate that the two most widely used cell types for promoting peripheral nerve regeneration may interact synergistically to aid their roles in peripheral nerve repair.