99 DIFFERENTIAL GENE REGULATION OF STEROIDOGENIC TRANSCRIPTS AND ESTRADIOL PRODUCTION FOLLOWING IN VITRO PIG EMBRYO ELONGATION IN ALGINATE HYDROGEL THREE-DIMENSIONAL MATRIX

2012 ◽  
Vol 24 (1) ◽  
pp. 162
Author(s):  
J. R. Miles ◽  
C. N. Sargus ◽  
S. A. Plautz ◽  
J. L. Vallet ◽  
A. K. Pannier
Keyword(s):  
Equal Opportunity ◽  
Culture Media ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Differential Gene Regulation ◽  
Alginate Hydrogels ◽  
The Media ◽  
And Control

Between Day 10 and 12 of gestation, the pig embryo elongates from a sphere to a long thin, filament. During this time, the embryo increases the production of oestrogen via an increase in steroidogenic transcripts, which is critical for maternal recognition of pregnancy. To date, attempts to elongate porcine embryos in vitro have been unsuccessful. Therefore, the objective of this study was to utilise alginate hydrogels to establish a culture system that promotes in vitro embryo elongation with a corresponding increase in steroidogenic transcripts and oestradiol production. In 3 replicate collections, White crossbred gilts (n = 15) were bred at Day 0 of the oestrous cycle. At Day 9 of gestation, reproductive tracts were collected and flushed with RPMI-1640 containing antibiotics. Embryos were recovered, grouped according to size and washed with RPMI-1640 containing antibiotics and 10% fetal bovine serum (FBS). Embryos were randomly assigned to be encapsulated using a double encapsulation technique (0.7% sodium alginate and 1.5% calcium chloride solution) or used as controls. Encapsulated and control embryos were cultured for 96 h in CO2 -pretreated RPMI-1640 containing antibiotics and 10% FBS at 38°C, 5% CO2 in air and 100% humidity. Every 24 h, the embryos were imaged and half of the media was replaced. The removed media was stored at –20°C and used to assess oestradiol levels by radioimmunoassay. At the end of culture, a subset of encapsulated and control embryos were snap frozen and used to assess the expression level of steroidogenic transcripts (STAR, CYP11 and CYP19) using quantitative PCR. All data were analysed using general linear model (GLM) procedures for ANOVA. Cell survival, assessed by blastocyst fragmentation and confirmed by live/dead staining in representative embryos, was greater (P = 0.01) for encapsulated embryos (60.1 ± 4.8%) compared with controls (33.3 ± 4.8%). Of encapsulated embryos, 27% had some morphological change (minor flattening and tubal formation) and 14% had significant morphological changes (considerable flattening and tubal formation elongating through the gel), consistent with in vivo embryo elongation. In contrast, the control embryos had no morphological changes observed and remained spherical during culture. The expression levels of STAR, CYP11 and CYP19 were significantly (P < 0.05) greater in encapsulated embryos compared with control embryos. Furthermore, a significant (P < 0.01) time-dependent increase in oestradiol levels in the culture media of encapsulated embryos was identified compared with controls and culture media alone. These results illustrate that cultured pig embryos encapsulated in alginate hydrogels undergo limited morphological changes with increased expression of steroidogenic transcripts and oestrogen production. †USDA is an equal opportunity provider and employer.

scholarly journals In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix

2014 ◽  
Vol 26 (7) ◽  
pp. 943 ◽  
Author(s):  
Catherine N. Sargus-Patino ◽  
Elane C. Wright ◽  
Sarah A. Plautz ◽  
Jeremy R. Miles ◽  
Jeff L. Vallet ◽  
...  
Keyword(s):  
Cell Survival ◽  
Culture System ◽  
Culture Media ◽  
Morphological Changes ◽  
Three Dimensional ◽  
In Vitro Development ◽  
Alginate Hydrogels ◽  
Vitro Development

Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 9, 10 or 11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96 h. Every 24 h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17β-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96 h in ENC and CONT embryos, as well as in vivo-developed control embryos (i.e. spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% (P < 0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P < 0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC–), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P < 0.001) in 17β-oestradiol was observed in culture media from ENC+ compared with ENC– and CONT embryos. These results illustrate that preimplantation pig embryos encapsulated in alginate hydrogels can undergo morphological changes with increased expression of steroidogenic transcripts and oestrogen production, consistent with in vivo-developed embryos. This alginate culture system can serve as a tool for evaluating specific mechanisms of embryo elongation that could be targeted to improve pregnancy outcomes.

scholarly journals Multifunctionalized hydrogels foster hNSC maturation in 3D cultures and neural regeneration in spinal cord injuries

2019 ◽  
Vol 116 (15) ◽  
pp. 7483-7492 ◽  
Author(s):  
Amanda Marchini ◽  
Andrea Raspa ◽  
Raffaele Pugliese ◽  
Marina Abd El Malek ◽  
Valentina Pastori ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injuries ◽  
Culture Media ◽  
Three Dimensional ◽  
Cholinergic Neurons ◽  
Good Manufacturing Practice ◽  
Neural Regeneration ◽  
Neuronal Markers

Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the “engineered tissue” was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.

scholarly journals Morphological Changes in Astrocytes by Self-Oxidation of Dopamine to Polydopamine and Quantification of Dopamine through Multivariate Regression Analysis of Polydopamine Images

Polymers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 2483
Author(s):  
Anik Karan ◽  
Elnaz Khezerlou ◽  
Farnaz Rezaei ◽  
Leon Iasemidis ◽  
Mark A. DeCoster
Keyword(s):  
Cell Culture ◽  
Regression Analysis ◽  
Multivariate Regression ◽  
Culture Media ◽  
Morphological Changes ◽  
Multivariate Regression Analysis ◽  
Cell Number ◽  
Polymerization Process

Astrocytes, also known as astroglia, are important cells for the structural support of neurons as well as for biochemical balance in the central nervous system (CNS). In this study, the polymerization of dopamine (DA) to polydopamine (PDA) and its effect on astrocytes was investigated. The polymerization of DA, being directly proportional to the DA concentration, raises the prospect of detecting DA concentration from PDA optically using image-processing techniques. It was found here that DA, a naturally occurring neurotransmitter, significantly altered astrocyte cell number, morphology, and metabolism, compared to astrocytes in the absence of DA. Along with these effects on astrocytes, the polymerization of DA to PDA was tracked optically in the same cell culture wells. This polymerization process led to a unique methodology based on multivariate regression analysis that quantified the concentration of DA from optical images of astrocyte cell culture media. Therefore, this developed methodology, combined with conventional imaging equipment, could be used in place of high-end and expensive analytical chemistry instruments, such as spectrophotometry, mass spectrometry, and fluorescence techniques, for quantification of the concentration of DA after polymerization to PDA under in vitro and potentially in vivo conditions.

scholarly journals cAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells

2010 ◽  
Vol 298 (3) ◽  
pp. F643-F654 ◽  
Author(s):  
Teodor G. Păunescu ◽  
Marija Ljubojevic ◽  
Leileata M. Russo ◽  
Christian Winter ◽  
Margaret M. McLaughlin ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Morphological Changes ◽  
Collecting Duct ◽  
Three Dimensional ◽  
Intercalated Cells ◽  
Proton Extrusion ◽  
Dependent Growth ◽  
Ratiometric Probe

Kidney proton-secreting A-intercalated cells (A-IC) respond to systemic acidosis by accumulating the vacuolar ATPase (V-ATPase) in their apical membrane and by increasing the length and number of apical microvilli. We show here that the cell-permeant cAMP analog CPT-cAMP, infused in vivo, results in an almost twofold increase in apical V-ATPase accumulation in AE1-positive A-IC within 15 min and that these cells develop an extensive array of apical microvilli compared with controls. In contrast, no significant change in V-ATPase distribution could be detected by immunocytochemistry in B-intercalated cells at the acute time point examined. To show a direct effect of cAMP on A-IC, we prepared cell suspensions from the medulla of transgenic mice expressing EGFP in IC (driven by the B1-subunit promoter of the V-ATPase) and exposed them to cAMP analogs in vitro. Three-dimensional reconstructions of confocal images revealed that cAMP induced a time-dependent growth of apical microvilli, starting within minutes after addition. This effect was blocked by the PKA inhibitor myristoylated PKI. These morphological changes were paralleled by increased cAMP-mediated proton extrusion (pHi recovery) by A-IC in outer medullary collecting ducts measured using the ratiometric probe BCECF. These results, and our prior data showing that the bicarbonate-stimulated soluble adenylyl cyclase (sAC) is highly expressed in kidney intercalated cells, support the idea that cAMP generated either by sAC, or by activation of other signaling pathways, is part of the signal transduction mechanism involved in acid-base sensing and V-ATPase membrane trafficking in kidney intercalated cells.

Improved Functional Survival of Human Islets of Langerhans in Three-Dimensional Matrix Culture

1994 ◽  
Vol 3 (5) ◽  
pp. 427-435 ◽  
Author(s):  
Mathias D. Brendel ◽  
Shen Shen Kong ◽  
Rodolfo Alejandro ◽  
Daniel H. Mintz
Keyword(s):  
Culture Media ◽  
Three Dimensional ◽  
Culture Method ◽  
Threshold Level ◽  
Insulin Dependent Diabetes Mellitus ◽  
Absolute Number ◽  
Human Islets ◽  
Dependent Diabetes Mellitus

The current study evaluates functional survival of human islets maintained in tissue culture for up to 4 wk in suspension media (CMRL-1066 with supplements) and contrasts these results with immobilizing three-dimensional matrices (agarose or alginate). The absolute number and volume of islets retrieved from agarose is significantly higher after two and four wk of culture compared to conventional free-floating media. In vitro function of islets, assessed by insulin/DNA content, insulin secretion into the culture media over 24 h and glucose-theophylline stimulated insulin release in a dynamic perifusion system, was not significantly different between free-floating and matrix preserved islets. In vivo islet function was evaluated by the effectiveness for reversal of insulin-dependent diabetes mellitus by transplantation of the islets under the kidney capsule of nude mice. Although adequate insulin responses to glucose were seen after culture in conventional or matrix media, only agarose embedded islets were consistently able to induce normoglycemia in diabetic recipients after 14 days of culture. Additional transplantation experiments defined the threshold level required to reverse diabetes to be between 1,000 and 1,500 agarose preserved islets. Our data suggest improved engraftment of human islets after agarose culture. This culture method may be of benefit for the accumulation of functionally competent human islets, thus facilitating the implementation of clinical protocols that utilize freshly isolated islets from multiple donors without the need for cryopreservation.

The impact of distinct culture media inLeishmania infantumbiology and infectivity

Parasitology ◽  
2013 ◽  
Vol 141 (2) ◽  
pp. 192-205 ◽  
Author(s):  
NUNO SANTARÉM ◽  
JOANA CUNHA ◽  
RICARDO SILVESTRE ◽  
CÁTIA SILVA ◽  
DIANA MOREIRA ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Culture Media ◽  
Rational Approach ◽  
Cycle Progression ◽  
The Media ◽  
Parasite Biology ◽  
Basic Characteristics ◽  
The Impact

SUMMARYAn ideal culture medium forLeishmaniapromastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinctin vitroandin vivoinfectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for thein vitromaintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

scholarly journals Modulation of the morphology and glycosaminoglycan biosynthesis of human monocytes, induced by culture substrates

1984 ◽  
Vol 219 (3) ◽  
pp. 793-799 ◽  
Author(s):  
S O Kolset ◽  
R Seljelid ◽  
U Lindahl
Keyword(s):  
Foreign Body ◽  
Culture Media ◽  
Chondroitin Sulphate ◽  
Morphological Changes ◽  
Artificial Substrates ◽  
Plastic Substrate ◽  
Incubation Periods ◽  
Cells Cultured

Monocytes were isolated from human blood and cultured in vitro on plastic culture dishes or on fibronectin-coated dishes. After 5 days in vitro, the cells on plastic dishes displayed marked morphological changes compared with day 1, with an epithelioid appearance resembling that of foreign-body cells. This transition was inhibited in cells cultured on fibronectin-coated dishes. 35S-labelled polysaccharides were isolated from the culture media after 24h incubation periods with inorganic [35S]sulphate. The cells cultured for 5 days on a plastic substrate synthesized, and secreted into the medium, an oversulphated galactosaminoglycan previously shown to contain 4,6-di-O-sulphated N-acetylgalactosamine units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. In contrast, 35S-labelled polysaccharide produced by cells cultured on plastic for 1 day only, or on fibronectin for either 1 or 5 days, contained only minor amounts of such disulphated sugar units. These findings indicate that the formation of oversulphated chondroitin sulphate is coupled to the conversion of monocytes into epithelioid cells. Furthermore, they suggest that the overall process is induced by contact with artificial substrates, and that it may be regarded as the equivalent of a foreign-body reaction in vivo.

scholarly journals Structure-based Design of CDC42 Effector Interaction Inhibitors For the Treatment of Cancer

2021 ◽  
Author(s):  
Sohail Jahid ◽  
Jose A. Ortega ◽  
Linh M. Vuong ◽  
Isabella Maria Acquistapace ◽  
Stephanie J. Hachey ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Three Dimensional ◽  
Pharmacokinetic Profile ◽  
Computer Aided Drug Design ◽  
New Class ◽  
Downstream Effectors ◽  
And Control ◽  
Braf Mutant

CDC42 family GTPases (RHOJ, RHOQ, CDC42) are upregulated but rarely mutated in cancer and control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. Here we use computer-aided drug design to discover a new chemical entity (ARN22089) that targets CDC42 GTPases and blocks CDC42 effector interactions without affecting the binding between closely related GTPases (RAC1, RAS, RAL) and their downstream effectors. Our lead compound has broad activity against a panel of cancer cell lines, inhibits S6 phosphorylation and MAPK activation, activates pro-inflammatory and apoptotic signaling, and blocks tumor growth and angiogenesis in three-dimensional vascularized microtumor models (VMT) in vitro. In addition, ARN22089 has a favorable pharmacokinetic profile and can inhibit the growth of BRAF mutant mouse melanomas and patient-derived xenografts in vivo. Taken together, this work identifies a promising new class of therapeutic agents that influence tumor growth by modulating CDC42 signaling in both the tumor cell and its microenvironment.

scholarly journals Development and Application of an Additively Manufactured Calcium Chloride Nebulizer for Alginate 3D-Bioprinting Purposes

2018 ◽  
Vol 9 (4) ◽  
pp. 63 ◽  
Author(s):  
Lukas Raddatz ◽  
Antonina Lavrentieva ◽  
Iliyana Pepelanova ◽  
Janina Bahnemann ◽  
Dominik Geier ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Media ◽  
Single Cells ◽  
Matrix Material ◽  
Three Dimensional ◽  
Culture Cell ◽  
3D Bioprinting ◽  
Cacl2 Solution

Three-dimensional (3D)-bioprinting enables scientists to mimic in vivo micro-environments and to perform in vitro cell experiments under more physiological conditions than is possible with conventional two-dimensional (2D) cell culture. Cell-laden biomaterials (bioinks) are precisely processed to bioengineer tissue three-dimensionally. One primarily used matrix material is sodium alginate. This natural biopolymer provides both fine mechanical properties when gelated and high biocompatibility. Commonly, alginate is 3D bioprinted using extrusion based devices. The gelation reaction is hereby induced by a CaCl2 solution in the building chamber after material extrusion. This established technique has two main disadvantages: (1) CaCl2 can have toxic effects on the cell-laden hydrogels by oxygen diffusion limitation and (2) good printing resolution in the CaCl2 solution is hard to achieve, since the solution needs to be removed afterwards and substituted by cell culture media. Here, we show an innovative approach of alginate bioprinting based on a CaCl2 nebulizer. The device provides CaCl2 mist to the building platform inducing the gelation. The necessary amount of CaCl2 could be decreased as compared to previous gelation strategies and limitation of oxygen transfer during bioprinting can be reduced. The device was manufactured using the MJP-3D printing technique. Subsequently, its digital blueprint (CAD file) can be modified and additive manufactured easily and mounted in various extrusion bioprinters. With our approach, a concept for a more gentle 3D Bioprinting method could be shown. We demonstrated that the concept of an ultrasound-based nebulizer for CaCl2 mist generation can be used for 3D bioprinting and that the mist-induced polymerization of alginate hydrogels of different concentrations is feasible. Furthermore, different cell-laden alginate concentrations could be used: Cell spheroids (mesenchymal stem cells) and single cells (mouse fibroblasts) were successfully 3D printed yielding viable cells and stable hydrogels after 24 h cultivation. We suggest our work to show a different and novel approach on alginate bioprinting, which could be useful in generating cell-laden hydrogel constructs for e.g., drug screening or (soft) tissue engineering applications.

scholarly journals Self-organization of organoids from endoderm-derived cells

2020 ◽  
Author(s):  
Allison Lewis ◽  
Rashmiparvathi Keshara ◽  
Yung Hae Kim ◽  
Anne Grapin-Botton
Keyword(s):  
Three Dimensional ◽  
Cellular Level ◽  
Self Organization ◽  
In Vivo Models ◽  
Differentiated Cells ◽  
Starting Conditions ◽  
External Conditions ◽  
The Media

AbstractOrganoids constitute biological systems which are used to model organ development, homeostasis, regeneration, and disease in vitro and hold promise for use in therapy. Reflecting in vivo development, organoids form from tissue cells or pluripotent stem cells. Cues provided from the media and individual cells promote self-organization of these uniform starting cells into a structure, with emergent differentiated cells, morphology, and often functionality that resemble the tissue of origin. Therefore, organoids provide a complement to two-dimensional in vitro culture and in vivo animal models of development, providing the experimental control and flexibility of in vitro methods with the three-dimensional context of in vivo models, with fewer ethical restraints than human or animal work. However, using organoids, we are only just beginning to understand on the cellular level how the external conditions and signaling between individual cells promote the emergence of cells and structures. In this review, we focus specifically on organoids derived from endodermal tissues: the starting conditions of the cells, signaling mechanisms, and external media that allow the emergence of higher order self-organization.

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