Expansion of Human Pluripotent Stem Cell-derived Early Cardiovascular Progenitor Cells by a Cocktail of Signaling Factors

Abstract Cardiovascular progenitor cells (CPCs) derived from human pluripotent stem cells (hPSCs) are proposed to be invaluable cell sources for experimental and clinical studies. This wide range of applications necessitates large-scale production of CPCs in an in vitro culture system, which enables both expansion and maintenance of these cells. In this study, we aimed to develop a defined and efficient culture medium that uses signaling factors for large-scale expansion of early CPCs, called cardiogenic mesodermal cells (CMCs), which were derived from hPSCs. Chemical screening resulted in a medium that contained a reproducible combination of three factors (A83-01, bFGF, and CHIR99021) that generated 1014 CMCs after 10 passages without the propensity for tumorigenicity. Expanded CMCs retained their gene expression pattern, chromosomal stability, and differentiation tendency through several passages and showed both the safety and possible cardio-protective potentials when transplanted into the infarcted rat myocardium. These CMCs were efficiently cryopreserved for an extended period of time. This culture medium could be used for both adherent and suspension culture conditions, for which the latter is required for large-scale CMC production. Taken together, hPSC-derived CMCs exhibited self-renewal capacity in our simple, reproducible, and defined medium. These cells might ultimately be potential, promising cell sources for cardiovascular studies.

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Biochemical and morpho-anatomical analyses of strawberry vitroplants hyperhydric tissues affected by BA and gelling agents

Revista CERES ◽

10.1590/s0034-737x2013000200002 ◽

2013 ◽

Vol 60 (2) ◽

pp. 152-160 ◽

Cited By ~ 5

Author(s):

Leticia Mascarenhas Pereira Barbosa ◽

Vespasiano Borges de Paiva Neto ◽

Leonardo Lucas Carnevalli Dias ◽

Reginaldo Alves Festucci-Buselli ◽

Rodrigo Sobreira Alexandre ◽

...

Keyword(s):

In Vitro Propagation ◽

Large Scale ◽

Culture Medium ◽

Shoot Proliferation ◽

Cellular Level ◽

Fragaria X Ananassa ◽

Scale Production ◽

Ms Medium ◽

Large Scale Production

In vitro propagation has become an effective practice for large-scale production of strawberry plants. The objective of this study was to evaluate the hyperhydricity and the multiplication capacity of two strawberry varieties (Fragaria x ananassa Duch. 'Dover' and 'Burkley') propagated in vitro. Plants maintained in MS medium supplemented with 1.0 mg L-1 BA were individualized and transferred to the same medium solidified with Agar (6.5 g L-1) or Phytagel® (2.5 g L-1) and BA at different concentrations (0; 0.5; 1.0; 2.0 and 3.0 mg L-1). Biochemical and anatomical analyses were carried out, as well as the analysis of the morphological hyperhydricity characteristics. The analysis of data showed: a) the increase in cytokinin concentration increased hyperhydricity frequency in both varieties; b) at concentrations up to 2.0 mg L-1 BA, the replacement of Agar by Phytagel® induced a higher formation of hyperhydric shoots; and c) the addition of BA induced oxidative stress, which is characterized by increased antioxidant activity and lipid peroxidation, as well as alterations at the cellular level, such as malformation of stomata and epidermal cells. In conclusion, the culture medium containing 0.5 mg L-1 BA solidified with Agar provided lower hyperhydricity percentages in association with higher rates of shoot proliferation in strawberry.

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In vitro culture and diversity of endophytic fungi in Bambusa oldhamii

Pesquisa Agropecuária Tropical ◽

10.1590/1983-40632019v4953760 ◽

2019 ◽

Vol 49 ◽

Cited By ~ 1

Author(s):

Ana Paula de Azevedo Pasqualini ◽

Mariely Cristine dos Santos ◽

Bruno Francisco Sant´Anna-Santos ◽

Hugo Pacheco de Freitas Fraga ◽

Marguerite Quoirin

Keyword(s):

In Vitro Culture ◽

Molecular Identification ◽

Endophytic Fungi ◽

Large Scale ◽

Culture Medium ◽

Current Knowledge ◽

Nodal Segments ◽

Scale Production ◽

Bambusa Oldhamii

ABSTRACT Bambusa oldhamii Munro is a fast-growing species of woody bamboo with strong commercial appeal. In Brazil, the use of this species is limited, mainly due to the low availability of seedlings for commercial plantations. Micropropagation is a technique used for the large scale production of seedlings, but protocols for the establishment of aseptic cultures are hampered by the presence of endophytic contamination. This study aimed to develop an in vitro establishment protocol for B. oldhamii, as well as to make the molecular identification of fungi associated with the explants used. Nodal segments of adult plants grown in the field were used as explants. This material was submitted to two experiments carried out to evaluate the effect of 6-benzylaminopurine (BAP) on multiplication and of Plant Preservative Mixture (PPMTM) as a disinfectant. In the first one, 10 µM, 15 µM or 20 µM of BAP were combined with 1 mL L-1, 2 mL L-1 or 3 mL L-1 of PPMTM; while the second one used 0 µM, 2.5 µM, 5 µM or 7.5 µM of BAP with 4 mL L-1 of PPMTM, both added to MS culture medium. After 21 days of culture, the use of 4 mL L-1 of PPMTM inhibited the bacterial growth and reduced fungal contamination. The addition of BAP to the culture medium above 10 µM inhibited the formation and growth of new shoots, while additions of less than 7,5 µM had no effect. The molecular identification of the endophytic fungi isolated during the in vitro culture indicated the presence of numerous fungal species, increasing the current knowledge about the diversity of fungi associated with bamboo.

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A Chemically Defined, Xeno- and Blood-Free Culture Medium Sustains Increased Production of Small Extracellular Vesicles From Mesenchymal Stem Cells

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.619930 ◽

2021 ◽

Vol 9 ◽

Author(s):

Aliosha I. Figueroa-Valdés ◽

Catalina de la Fuente ◽

Yessia Hidalgo ◽

Ana María Vega-Letter ◽

Rafael Tapia-Limonchi ◽

...

Keyword(s):

Extracellular Vesicles ◽

Large Scale ◽

Culture Medium ◽

Culture Media ◽

Cell Phenotype ◽

Target Cells ◽

Scale Production ◽

Paracrine Factors

Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV’s secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV’s production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV’s characteristics and functionality and the parental cell’s phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method’s interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.

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Composição do meio de cultivo para produção de microplantas de caju-de-árvore-do-Cerrado (Anacardium othonianum RIZZ.) - Composition of the cultivation medium for the production of microplants of Cerrado-tree cashew (Anacardium othonianum RIZZ.)

Científic - Multidisciplinary Journal ◽

10.29247/2358-260x.2017v4i1.p01-11 ◽

2017 ◽

Vol 4 (1) ◽

pp. 01 ◽

Cited By ~ 1

Author(s):

Ísis Danielle Sousa ◽

Jackellyne Bruna Sousa ◽

Flávia Dionísio Pereira ◽

João Das Graças Santana ◽

Aurélio Rubio Neto ◽

...

Keyword(s):

Activated Charcoal ◽

Large Scale ◽

Culture Medium ◽

Average Length ◽

Scale Production ◽

Cultivation Medium ◽

Large Scale Production ◽

Number Of Leaves ◽

Short Time

RESUMOO Anacardium othonianum Rizz. é uma planta típica de regiões de clima tropical caracterizada pela aparência exótica e aroma agradável. Na busca de diversificar a produção e atividades que proporcione maior rentabilidade, a micropropagação tem sido uma alternativa para produção em grande escala em curto espaço de tempo. Por isso, objetivou-se com este trabalho determinar as melhores condições in vitro para micropropagação dessa espécie, para isso, avaliamos a adição de diferentes concentrações de AIB (Ácido Indolbutírico), sacarose e carvão ativado no meio de cultivo in vitro. Foram utilizadas cinco concentrações de AIB (0; 1; 2; 3; 4 mg L-1) e cinco concentrações de sacarose (0, 15, 30, 45, 60 g L-1) na ausência ou presença de carvão ativado (2 g L-1), em meio WPM 50%. Aos 30 e 60 dias foram feitas avaliações do número de explantes oxidados, comprimento médio e número de folhas por explante. Verificou-se que o meio de cultivo suplementado com carvão ativado e a adição de 4 mg L-1 de AIB, contribuiu para a o crescimento de raízes in vitro da espécie. Enquanto, que o meio de cultivo com 30 g sacarose e presença de carvão ativado proporcionou maior comprimento dos explantes e maior número de folhas.Palavras-chave: Frutífera nativa, cerrado, micropropagação.ABSTRACTThe Anacardium othonianum Rizz. It’s a typical plant of regions of tropical climate characterized by the exotic appearance and pleasant aroma. In the quest to diversify production and activities that provide greatest profitability, micropropagation has been an alternative for large-scale production in short time. Therefore, the objective of this work is to determine the best in vitro results for micropropagation of this species, for this, we evaluated the addition of different concentrations of AIB (Indolbutyric Acid), sucrose and activated charcoal in the in vitro culture medium. Five concentrations of AIB (0, 1, 2, 3, 4 mg L-1) and five sucrose concentrations (0, 15, 30, 45, 60 g L-1) were used in the absence or presence of activated charcoal, in WPM 50% medium. At 30 and 60 days, the number of oxidation, average length and number of leaves per explant were evaluated. It was found that the culture medium supplemented with activated charcoal and an addition of 4 mg L-1 of IBA, contributed to in vitro root growth. While the culture medium with 30 g L-1 sucrose and the presence of activated charcoal provided a longer length of the explants and a larger number of leaves.Key words: Native Fruit, Cerrado, Micropropagation.

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Addressing Challenges to Enhance the Bioactives ofWithania somniferathrough Organ, Tissue, and Cell Culture Based Approaches

BioMed Research International ◽

10.1155/2017/3278494 ◽

2017 ◽

Vol 2017 ◽

pp. 1-15 ◽

Cited By ~ 4

Author(s):

Pritika Singh ◽

Rupam Guleri ◽

Amrita Angurala ◽

Kuldeep Kaur ◽

Kulwinder Kaur ◽

...

Keyword(s):

Cell Culture ◽

Secondary Metabolites ◽

Culture System ◽

Large Scale ◽

Distinct Advantage ◽

Scale Production ◽

Genetic Base ◽

Wide Range ◽

Organ Tissue

Withania somniferais a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues.In vitropropagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential ofin vitrocell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years.In vitroculture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.

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Human Paraoxonase 1 as a Pharmacologic Agent: Limitations and Perspectives

The Scientific World JOURNAL ◽

10.1155/2014/854391 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 6

Author(s):

Priyanka Bajaj ◽

Rajan K. Tripathy ◽

Geetika Aggarwal ◽

Abhay H. Pande

Keyword(s):

Large Scale ◽

Ex Vivo ◽

Expression System ◽

Active Form ◽

Hydrolytic Activity ◽

Paraoxonase 1 ◽

Scale Production ◽

Pharmacologic Agent ◽

Wide Range

Human PON1 (h-PON1) is a multifaceted enzyme and can hydrolyze (and inactivate) a wide range of substrates. The enzyme shows anti-inflammatory, antioxidative, antiatherogenic, ant-diabetic, antimicrobial, and organophosphate (OP)-detoxifying properties. However, there are certain limitations regarding large-scale production and use of h-PON1 as a therapeutic candidate. These include difficulties in producing recombinant h-PON1 (rh-PON1) using microbial expression system, low hydrolytic activity of wild-type h-PON1 towards certain substrates, and low storage stability of the purified enzyme. This review summarizes the work done in our laboratory to address these limitations. Our results show that (a) optimized polynucleotide sequence encoding rh-PON1 can express the protein in an active form inE. coliand can be used to generate variant of the enzyme having enhanced hydrolytic activity, (b)in vitrorefolding of rh-PON1 enzyme can dramatically increase the yield of an active enzyme, (c) common excipients can be used to stabilize purified rh-PON1 enzyme when stored under different storage conditions, and (d) variants of rh-PON1 enzyme impart significant protection against OP-poisoning in human blood (ex vivo) and mouse (in vivo) model of OP-poisoning. The rh-PON1 variants and their process of production discussed here will help to develop h-PON1 as a therapeutic candidate.

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Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽

10.3390/v13071288 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1288

Author(s):

Wendy Dong ◽

Boris Kantor

Keyword(s):

Large Scale ◽

Lentiviral Vector ◽

Clinical Applications ◽

Scale Production ◽

Base Editing ◽

Epigenome Editing ◽

Vector Systems ◽

Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

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Xylanolytic Bacillus species for xylooligosaccharides production: a critical review

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00369-3 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Rozina Rashid ◽

Muhammad Sohail

Keyword(s):

Large Scale ◽

Extracellular Enzymes ◽

Membrane Separation ◽

Well Being ◽

Bacillus Species ◽

Scale Production ◽

Food Ingredients ◽

Large Scale Production ◽

Wide Range ◽

Biological Impacts

AbstractThe capacity of different Bacillus species to produce large amounts of extracellular enzymes and ability to ferment various substrates at a wide range of pH and temperature has placed them among the most promising hosts for the industrial production of many improved and novel products. The global interest in prebiotics, for example, xylooligosaccharides (XOs) is ever increasing, rousing the quest for various forms with expanded productivity. This article provides an overview of xylanase producing bacilli, with more emphasis on their capacity to be used in the production of the XOs, followed by the purification strategies, characteristics and application of XOs from bacilli. The large-scale production of XOs is carried out from a number of xylan-rich lignocellulosic materials by chemical or enzymatic hydrolysis followed by purification through chromatography, vacuum evaporation, solvent extraction or membrane separation methods. Utilization of XOs in the production of functional products as food ingredients brings well-being to individuals by improving defense system and eliminating pathogens. In addition to the effects related to health, a variety of other biological impacts have also been discussed.

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A modular microfluidic system based on a multilayered configuration to generate large-scale perfusable microvascular networks

Microsystems & Nanoengineering ◽

10.1038/s41378-020-00229-8 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Tao Yue ◽

Da Zhao ◽

Duc T. T. Phan ◽

Xiaolin Wang ◽

Joshua Jonghyun Park ◽

...

Keyword(s):

Large Scale ◽

Circulatory System ◽

Vertical Direction ◽

Microfluidic System ◽

Vital Role ◽

Microvascular Networks ◽

Biological Studies ◽

Wide Range ◽

High Flexibility

AbstractThe vascular network of the circulatory system plays a vital role in maintaining homeostasis in the human body. In this paper, a novel modular microfluidic system with a vertical two-layered configuration is developed to generate large-scale perfused microvascular networks in vitro. The two-layer polydimethylsiloxane (PDMS) configuration allows the tissue chambers and medium channels not only to be designed and fabricated independently but also to be aligned and bonded accordingly. This method can produce a modular microfluidic system that has high flexibility and scalability to design an integrated platform with multiple perfused vascularized tissues with high densities. The medium channel was designed with a rhombic shape and fabricated to be semiclosed to form a capillary burst valve in the vertical direction, serving as the interface between the medium channels and tissue chambers. Angiogenesis and anastomosis at the vertical interface were successfully achieved by using different combinations of tissue chambers and medium channels. Various large-scale microvascular networks were generated and quantified in terms of vessel length and density. Minimal leakage of the perfused 70-kDa FITC-dextran confirmed the lumenization of the microvascular networks and the formation of tight vertical interconnections between the microvascular networks and medium channels in different structural layers. This platform enables the culturing of interconnected, large-scale perfused vascularized tissue networks with high density and scalability for a wide range of multiorgan-on-a-chip applications, including basic biological studies and drug screening.

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