REVIEW ON IN-VITRO AMEBOCYTE CULTURE–A LESSON LEARNED FROM PAST

2015 ◽  
Vol 77 (25) ◽  
Author(s):  
Hassan I. Sheikh ◽  
Akbar John, B. ◽  
Solachuddin J. A. Ichwan ◽  
Zaleha, K. ◽  
Kamaruzzaman, B. Y.
Keyword(s):  
Culture Media ◽  
Sole Source ◽  
Culture Conditions ◽  
Standard Test ◽  
Wild Stock ◽  
Implantable Medical Devices ◽  
Current Form ◽  
Horseshoe Crabs ◽  
A Cell

Limulus Amebocyte Lysate (LAL/TAL) assay is the only standard test approved by the Food and Drug Administration (FDA) to quantify bacterial endotoxin in injectable drugs and implantable medical devices. At present, horseshoe crabs are the sole source of LAL/TAL. Biomedical companies harvest and bleed horseshoe crabs which lead to ≤30% post bleeding mortality. The continuity of this practices will eventually deplete wild stock and threat horseshoe crab’s population. Though, many alternative biosensors were developed to quantify endotoxins, they are not without limitations and some of them even need LAL/TAL as source detectors. Hence, the LAL/TAL industry in its current form has ethical, ecological, commercial and technical issues that make it unideal standard test. An alternative method of culturing amebocyte in tissue culture medium has been addressed in literature since last 4 decades. This paper will address the issues in amebocyte culture in-vitro based on the published literatures. The paper will also suggest the best source of amebocyte cells (gill, gill flaps, blood), culture mode (monolayer or suspension), culture media (Grace's Insect Medium, Leibovitz’s L-15 Medium, Modified Essential medium, Shields and Sang insect medium, TNM-FH Medium, RPMI 1640 medium) and best culture conditions (pH, temperature, osmolarity). This review will also emphasize on key elements in establishing a cell line for the continuous harvesting of amebocyte in-vitro.

scholarly journals Presence of LH receptor mRNA in granulosa cells as a potential marker of oocyte developmental competence and characterization of the bovine splicing isoforms

Reproduction ◽  
2003 ◽  
pp. 437-446 ◽  
Author(s):  
C Robert ◽  
D Gagne ◽  
JG Lussier ◽  
D Bousquet ◽  
FL Barnes ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Culture Media ◽  
Protein Product ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Lh Receptor ◽  
Potential Marker ◽  
A Cell

As the expression of the LH receptor (LH-R) in granulosa cells is thought to be associated with later stages of folliculogenesis, this study was undertaken to evaluate the presence of LH-R mRNA as a suitable marker for developmental competence of oocytes. Granulosa cells and cumulus-oocyte complexes (COCs) were recovered from cows that had received ovarian stimulation. The COCs were subjected to embryo production procedures in vitro to assess the embryonic potential of the oocyte, and the corresponding granulosa cells were used to evaluate the presence of LH-R mRNA by RT-PCR. The presence of LH-R transcripts in granulosa cells is not a key characteristic of a follicle bearing a competent oocyte, although a higher proportion of oocytes reach the blastocyst stage when LH-R mRNA is detected in the granulosa cells. Different LH-R isoforms were cloned and sequence discrepancies among six of the isoforms enabled the design of specific oligonucleotides to study the presence of the isoforms in different follicular cells. All LH-R transcripts studied and the 80 kDa protein product corresponding to the full length receptor were found in granulosa cells of small (< 4 mm) and large (> 5 mm) follicles. When the granulosa cells were cultured, the transcripts were downregulated by the culture conditions; downregulation was more acute in granulosa cells from small follicles. The addition of LH to the culture media enhanced LH-R mRNA downregulation. The presence of several LH-R transcript isoforms was tissue specific and in the theca cells LH-R mRNA was restricted mainly to cells from larger follicles. This finding indicates that the expression and the splicing of LH-R mRNA are regulated in a cell-specific and follicular size-specific manner.

Biophysical stimulation for in vitro engineering of functional cardiac tissues

2017 ◽  
Vol 131 (13) ◽  
pp. 1393-1404 ◽  
Author(s):  
Anastasia Korolj ◽  
Erika Yan Wang ◽  
Robert A. Civitarese ◽  
Milica Radisic
Keyword(s):  
Cardiac Tissue ◽  
Culture Media ◽  
Culture Conditions ◽  
Lineage Specification ◽  
Cardiac Tissues ◽  
Cardiac Lineage ◽  
And Function ◽  
Tissue Models

Engineering functional cardiac tissues remains an ongoing significant challenge due to the complexity of the native environment. However, our growing understanding of key parameters of the in vivo cardiac microenvironment and our ability to replicate those parameters in vitro are resulting in the development of increasingly sophisticated models of engineered cardiac tissues (ECT). This review examines some of the most relevant parameters that may be applied in culture leading to higher fidelity cardiac tissue models. These include the biochemical composition of culture media and cardiac lineage specification, co-culture conditions, electrical and mechanical stimulation, and the application of hydrogels, various biomaterials, and scaffolds. The review will also summarize some of the recent functional human tissue models that have been developed for in vivo and in vitro applications. Ultimately, the creation of sophisticated ECT that replicate native structure and function will be instrumental in advancing cell-based therapeutics and in providing advanced models for drug discovery and testing.

The effect of osmolarity on mouse parthenogenesis

Development ◽  
1974 ◽  
Vol 31 (2) ◽  
pp. 513-526
Author(s):  
M. H. Kaufman ◽  
M. A. H. Surani
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Culture Media ◽  
Polar Body ◽  
Amino Acid Mixture ◽  
Culture Conditions ◽  
Follicle Cells ◽  
Acid Mixture ◽  
Second Polar Body

Eggs from (C57B1 × A2G)F1 mice were activated by treatment with hyaluronidase, which removed the follicle cells, and cultured in vitro. Observations were made 6–8 h after hyaluronidase treatment to determine the frequency of activation and the types of parthenogenones induced. Cumulus-free eggs resulting from hyaluronidase treatment were incubated for 2¼ h in culture media of various osmolarities. The frequency of activation was found to be dependent on the postovulatory age of oocytes, while the types of parthenogenones induced were dependent on the osmolarity of the in vitro culture medium and their postovulatory age. Culture in low osmolar medium suppressed the extrusion of the second polar body (2PB). This decreased the incidence of haploid eggs with a single pronucleus and 2PB and immediately cleaved eggs from 97·5% to 42·3% of the activated population. Where 2PB extrusion had been suppressed, 97·4% of parthenogenones contained two haploid pronuclei. Very few were observed with a single and presumably diploid pronucleus. Serial observations from 11 to 18 h after hyaluronidase treatment were made on populations of activated eggs as they entered the first cleavage mitosis after 2¼ h incubation in medium either of normal (0·287 osmol) or low (0·168 osmol) osmolarity. A delay in the time of entry into the first cleavage mitosis similar to the duration of incubation in low osmolar medium was observed. Further, eggs were incubated in control and low osmolar culture media containing uniformly labelled [U-14C]amino acid mixture to examine the extent of protein synthesis in recently activated eggs subjected to these culture conditions. An hypothesis is presented to explain the effect of incubation in low osmolar culture medium in delaying the first cleavage mitosis.

scholarly journals Experimental Study on Cell-free Approach for Articular Cartilage Treatment

2019 ◽  
Vol 5 (1) ◽  
pp. 171-174 ◽  
Author(s):  
Gözde Dursun ◽  
Bernd Markert ◽  
Marcus Stoffel
Keyword(s):  
Articular Cartilage ◽  
Petri Dish ◽  
Treatment Method ◽  
Culture Conditions ◽  
Cyclic Compression ◽  
A Cell ◽  
Static Cultivation ◽  
Promising Treatment ◽  
Bioreactor System

AbstractCell-free based approaches are introduced as a promising treatment method for articular cartilage. The success of this method requires cell colonisation from resident tissue into cell-free implants. The objective of our study is to promote the cell colonisation into cell-free collagen I based implants by mechanical stimulation. Therefore, a new in vitro cellular model consisting chondrocyte-seeded matrix and cell-free implants was developed in a polydimethylsiloxan (PDMS) mold. These constructs were cultured under dynamic and static culture conditions. For the dynamic culture, we have developed an in-house bioreactor system where both the load and the deformation applied to the specimen are recorded. Cyclic compression, with a strain of 5% and frequency of 0.1 Hz, was applied to constructs without any break. At the end of three days of dynamic and static cultivation, the cell-free implants were seperated from cell-seeded matrix and cultured in a petri dish three days long. Afterwards, they were analysed using fluoresence dyes. The microscopic assessment indicated that there was a cell migration into the cell-free implants which were cultured dynamically.

187 INCREASED NUMBER OF EXPANDED BOVINE BLASTOCYSTS IN SOF AND CR1 RELATIVE TO KSOM

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. M. Kohl ◽  
R. L. Monson ◽  
L. E. Enwall ◽  
J. J. Rutledge
Keyword(s):  
Gene Expression ◽  
Culture Media ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Pregnancy Rates ◽  
Embryo Morphology ◽  
Bovine Embryos ◽  
Chi Square Analysis

Assessment of morphological stage grade is a subjective procedure. Stage grade is of vital importance to, among other things, recipient synchrony for the purpose of establishing successful pregnancies. Asynchronous embryo transfer has led to decreases in pregnancy rates (Farin et al. 1995 Biol. Reprod. 52, 676–682) and has been implicated in contributing to large offspring syndrome (Young et al. 1996 Theriogenology 45, 231). Differences in embryo kinetics based on culture conditions have been well documented (Mello et al. 2005 Reprod. Fert. Dev. 17, 221 abst). Whether such differences are the result of species, breed, metabolic stress, sire effects, or separation from an in vivo environment has yet to be determined. The correlation between oxygen respiration rates and embryo morphology as well as embryo diameter in bovine embryos produced in vitro has shown promise in the development of a more objective predictor of embryo quality and perhaps pregnancy initiation (Lopes et al. 2005 Reprod. Fert. Dev. 17, 151 abst). As well, recent examination of gene expression patterns of in vitro-derived bovine embryos seems to indicate that longer periods of in vitro culture are associated with lower rates of embryo survival (Lonergan et al. 2006 Theriogenology 65, 137–152). We hypothesize that differences do exist in the number, rate, and morphological appearance of blastocysts and that these parameters are in large part based on culture conditions in vitro. The objective of this experiment was to determine the timing and distribution of blastocyst formation of in vitro-produced bovine embryos cultured in SOF8, CR18AA, and KSOM8, under a standard incubation environment. Bovine ovaries from a local abattoir were aspirated and matured for 18-22. Oocytes were fertilized with frozen-thawed Percoll-separated semen from a Holstein bull. Presumptive zygotes were vortexed to remove cumulus cells and placed into 3 different culture media in a highly humidified atmosphere containing 20% oxygen, 5% carbon dioxide, and compressed air at 38.5�C. Embryos were evaluated specifically at 168 h post-insemination (Day 7) and assigned a morphological stage grade (IETS) to determine fixed time point differences. A total of 6 complete replicates were performed. Only embryos exhibiting the presence of a blastocoel at this time were documented (early blast, mid-blast, expanded blast). At 168 h post-insemination, there were no significant differences in the total number of embryos reaching early or mid-blast stage in any of the media. However, chi-square analysis revealed an increase in the number of expanded blastocysts in SOF (n = 813) and CR1 (n = 838) treatments compared to KSOM (n = 824; P &lt; 0.0001). Expanded blastocysts in SOF were also greater in number than in CR1 (P &lt; 0.05). Embryo selection based on development to the expanded blastocyst stage on Day 7 may prove useful in increasing pregnancy rates, and may validate qualitative correlations based on oxygen consumption and gene expression profiles for embryos produced in vitro.

Effect of culture conditions on artificial activation of porcine oocytes matured in vitro

1996 ◽  
Vol 8 (8) ◽  
pp. 1153 ◽  
Author(s):  
N Yamauchi ◽  
H Sasada ◽  
S Sugawara ◽  
T Nagai
Keyword(s):  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Treated Group ◽  
Culture Conditions ◽  
Culture Period ◽  
Subsequent Response ◽  
Artificial Activation

The effects of culture media used and culture period for in vitro maturation of porcine oocytes on their subsequent response to chemical and electrical activation, were investigated. Activated oocytes were identified by the presence of a pronucleus(ei) or cleavage. Porcine oocytes were cultured for 24, 30, 36, 42 and 48 h in TCM199 with Earle's salts (199) supplemented with 10% fetal calf serum (199-FCS) before electrical stimulation. Although few oocytes were activated after 24 h and 30 h of culture (5.4% and 6.1% respectively), the percentage of activated oocytes increased significantly to 93.2% after 42 h in culture (P < 0.05); however, when the culture period was extended to 48 h, there was a significant decrease to 56.7% (P < 0.05). Oocytes were also cultured in four types of media: (1) 199-FCS; (2) 199 supplemented with 5 mg mL-1 bovine serum albumin (199-BSA); (3) Kreb's-Ringer bicarbonate solution supplemented with 10% FCS (KRB-FCS); and (4) KRB supplemented with BSA (KRB-BSA). After 42 h of culture in each medium, the oocytes were electrically activated. Although rates of maturation of oocytes cultured in the four media were similar (74.0-80.8%), all oocytes except those cultured in 199-FCS failed to be activated. In addition, oocytes were cultured for 36, 42 and 48 h in 199-FCS and then stimulated by treatment with ethanol. Significantly fewer oocytes were activated in the chemically-treated group than in the electrically-treated group. These results indicate that culture conditions used for the culture of porcine oocytes in vitro are important with respect to their subsequent response to artificial activation.

In vitro culture of pointed gourd (Trichosanthes dioica Roxb.)

1970 ◽  
Vol 35 (1) ◽  
pp. 135-142 ◽  
Author(s):  
MA Malek ◽  
D Khanam ◽  
M Khatun ◽  
MH Molla ◽  
MA Mannan
Keyword(s):  
Plant Regeneration ◽  
In Vitro Culture ◽  
Culture Media ◽  
Culture Conditions ◽  
Root Induction ◽  
Ms Medium ◽  
Trichosanthes Dioica ◽  
Pointed Gourd ◽  
Regenerated Plantlets

An experiment was conducted to study the in vitro culture of pointed gourd. Cotyledon rescued from physiologically matured seeds (PMS) and immatured seeds (IMS) of pointed gourd were used as explants. Cotyledon excised from PMS responded very well in all culture conditions. Plant regenerated from cotyledon of PMS ranged from 38 to 96% in different hormonal formulations of culture media. Highest percentage of shoot regeneration was observed in MS + 1.0 mg/l BAP and lowest in MS + 2.5 mg/l BAP. No plant regeneration was observed in cotyledon from immatured seeds. The highest percentage of root induction (99%) was achieved in half MS medium supplemented with 0.5 mg/l NAA. The regenerated plantlets were successfully established in earthen pot. Keywords: Cotyledon; in vitro; pointed gourd. DOI: 10.3329/bjar.v35i1.5874Bangladesh J. Agril. Res. 35(1) : 135-142, March 2010

scholarly journals Commercial Biocontrol Agents Reveal Contrasting Comportments Against Two Mycotoxigenic Fungi in Cereals: Fusarium Graminearum and Fusarium Verticillioides

Toxins ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 152 ◽  
Author(s):  
Lucile Pellan ◽  
Noël Durand ◽  
Véronique Martinez ◽  
Angélique Fontana ◽  
Sabine Schorr-Galindo ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Culture Media ◽  
Fusarium Verticillioides ◽  
Inhibition Zone ◽  
Culture Conditions ◽  
Dual Culture ◽  
Growth Inhibition Zone ◽  
Mycotoxigenic Fungi ◽  
The Impact

The aim of this study was to investigate the impact of commercialized biological control agents (BCAs) against two major mycotoxigenic fungi in cereals, Fusarium graminearum and Fusarium verticillioides, which are trichothecene and fumonisin producers, respectively. With these objectives in mind, three commercial BCAs were selected with contrasting uses and microorganism types (T. asperellum, S. griseoviridis, P. oligandrum) and a culture medium was identified to develop an optimized dual culture bioassay method. Their comportment was examined in dual culture bioassay in vitro with both fusaria to determine growth and mycotoxin production kinetics. Antagonist activity and variable levels or patterns of mycotoxinogenesis inhibition were observed depending on the microorganism type of BCA or on the culture conditions (e.g., different nutritional sources), suggesting that contrasting biocontrol mechanisms are involved. S. griseoviridis leads to a growth inhibition zone where the pathogen mycelium structure is altered, suggesting the diffusion of antimicrobial compounds. In contrast, T. asperellum and P. oligandrum are able to grow faster than the pathogen. T. asperellum showed the capacity to degrade pathogenic mycelia, involving chitinolytic activities. In dual culture bioassay with F. graminearum, this BCA reduced the growth and mycotoxin concentration by 48% and 72%, respectively, and by 78% and 72% in dual culture bioassay against F. verticillioides. P. oligandrum progressed over the pathogen colony, suggesting a close type of interaction such as mycoparasitism, as confirmed by microscopic observation. In dual culture bioassay with F. graminearum, P. oligandrum reduced the growth and mycotoxin concentration by 79% and 93%, respectively. In the dual culture bioassay with F. verticillioides, P. oligandrum reduced the growth and mycotoxin concentration by 49% and 56%, respectively. In vitro dual culture bioassay with different culture media as well as the nutritional phenotyping of different microorganisms made it possible to explore the path of nutritional competition in order to explain part of the observed inhibition by BCAs.

Review: Role of tubal environment in preimplantation embryogenesis: application to co-culture assays

Zygote ◽  
2010 ◽  
Vol 19 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Pierre Guérin ◽  
Yves Ménézo
Keyword(s):  
Amino Acids ◽  
Embryo Culture ◽  
Metabolic Flux ◽  
Cultured Cells ◽  
Culture Media ◽  
Culture Conditions ◽  
Antioxidant Compounds ◽  
Beneficial Effects ◽  
Stage Embryo

SummaryThe culture of early preimplantation stage embryo is still delicate and the metabolic pathways of embryos are not completely understood. Embryo needs are evolutionary during the preimplantation development, consequently it is difficult to meet embryo needs in vitro. Culture conditions have to respect several physical and chemical equilibria: such as redox potential, pH, osmotic pressure, metabolic flux of energetic compounds, endogenous pools of amino acids and transcripts, etc. Embryo culture media are generally supplemented with amino acids, glucose, other energetic metabolites and antioxidant compounds, vitamin, and growth factors etc. Furthermore autocrine and paracrine regulation of embryo development probably exist. In fact embryo culture conditions have to be as non-toxic as possible. Various types of co-culture systems have been devised to overcome these problems. Complex interrelations exist between embryos and co-cultured cells. The beneficial effects of co-cultured cells may be due to continuous modifications of the culture medium, i.e. the elimination of toxic compounds and/or the supply of embryotrophic factors.

scholarly journals A KINETIC ANALYSIS OF MYOGENESIS IN VITRO

1972 ◽  
Vol 52 (1) ◽  
pp. 52-65 ◽  
Author(s):  
Michael C. O'Neill ◽  
Frank E. Stockdale
Keyword(s):  
Cell Cycle ◽  
Cell Fusion ◽  
Tritiated Thymidine ◽  
Culture Conditions ◽  
Complete Fusion ◽  
Low Density ◽  
A Cell ◽  
Muscle Cultures ◽  
S Period

Conditions which yielded reproducible growth kinetics with extensive, relatively synchronous differentiation are described for chick muscle cultures. The effects of cell density and medium changes on the timing of cell fusion were examined. Low-density cultures which received a change of medium at 24 hr after plating show the highest rate of cell fusion, increasing from 15 to 80% fused cells in a 10 hr period. These optimal culture conditions were employed to reexamine two questions from the earlier literature on muscle culture: (a) can cells which normally would fuse at the end of one cell cycle be forced to go through another cell cycle before fusion; and (b) how soon after its final S period can a cell complete fusion? In answer to the first question, it was found that if the medium is changed, many cells which would otherwise fuse can be made to undergo another cell cycle before fusion. In the second case, radioautographs were made from cultures incubated with tritiated thymidine for various times at the beginning of the fusion period. These show labeled nuclei in myotubes as early as 3 hr after the beginning of the incubation period. This indicates that cells can fuse as early as the beginning of the G1 period, and suggests that there is not an obligatory exit from the cell cycle or a prolonged G1 period before cell fusion and differentiation during myogenesis.

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