Can in vitro assays measure dead cells?
Cytotoxicity Assays: In Vitro Methods to Measure Dead Cells - Assay Guidance Manual - NCBI Bookshelf Membrane integrity is the feature most often used to detect whether eukaryotic cells cultured in vitro are alive or dead.
What is in vitro fertilization?
In vitro fertilization (IVF) Overview. In vitro fertilization (IVF) is a complex series of procedures used to treat fertility or genetic problems and assist with the conception of a child. During IVF, mature eggs are collected (retrieved) from your ovaries and fertilized by sperm in a lab.
What has changed in in vitro fertilization in the last decade?
Significant developments in the first decade led to greater efficiency and expanded accessibility of in vitro fertilization to the general public. In the ensuing decade, innovation and refinements in technology led to the introduction of ICSI, MESA, and TESE which provided effective treatment for male infertility.
What are the risks of in vitro fertilization?
Specific steps of an in vitro fertilization (IVF) cycle carry risks, including: Multiple births. Premature delivery and low birth weight. Ovarian hyperstimulation syndrome. Miscarriage. Egg-retrieval procedure complications. Ectopic pregnancy. Birth defects. Ovarian cancer. Stress.
What are in vitro studies?
In vitro is used to describe work that's performed outside of a living organism. This can include studying cells in culture or methods of testing the antibiotic sensitivity of bacteria.
What is cell line studies?
Cell lines have revolutionized scientific research and are being used in vaccine production, testing drug metabolism and cytotoxicity, antibody production, study of gene function, generation of artificial tissues (e.g., artificial skin) and synthesis of biological compounds e.g., therapeutic proteins.
What are cell line models?
Cell lines are in vitro model systems that are widely used in different fields of medical research, especially basic cancer research and drug discovery. Their usefulness is primarily linked to their ability to provide an indefinite source of biological material for experimental purposes.
What is an in vitro cell line?
Cell lines obtained from in vitro transformed cell lines or cancerous cells are indefinite cell lines and can be grown in monolayer or suspension form. These cells divide rapidly with a generation time of 12 to 14 hours and have a potential to be sub-cultured indefinitely.
What is cell culture system?
Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions.
What is cell culture technology?
Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro).
What does ATCC screen cell lines for?
ATCC human cells are invaluable for your public health research. Human cell lines are a critical biological resource. These in vitro models of human biology are used in biomedical research, toxicology studies, bioindustry, drug discovery, vaccine development, protein production, and a host of other applications.
Are cell culture and cell line same?
Primary cell culture is the culture of cells directly isolated from parental tissue of interest; whereas cell line is the culture of cells originated from a primary cell culture, which is generally used to expand cell population and prolong life span.
Which cell line uses biological research?
HEK293, or human embryonic kidney-derived epithelial cells, are arguably one of the most widely used cell lines in cell biology research.
How are drugs tested in vitro?
When testing drugs in vitro, the drug is confined to a known volume (ie, well of a plate, test tube, etc.), therefore the concentration of the drug is known. This is critical since all measures of drug activity (ie, potency, efficacy, affinity) are basically concentrations at which a defined drug effect is observed.
What does in vitro testing mean?
In vitro diagnostics (IVD) are tests done on samples such as blood or tissue that have been taken from the human body. In vitro diagnostics can detect diseases or other conditions, and can be used to monitor a person's overall health to help cure, treat, or prevent diseases.
What is MTT cytotoxicity assay?
Introduction. The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity.
What were the effects of assisted reproductive treatment on tubal infertility?
As the use of assisted reproductive treatments expanded from tubal infertility to include ovulation disorders, male factor infertility and decreased ovarian reserve, the number of ART cycles dramatically increased. The pioneering work reported by Steptoe and Edwards had required a surgical approach using laparoscopy. Subsequent modifications such as GIFT and ZIFT still relied upon laparoscopy. However, the necessity for general anesthesia and its attendant risks, along with the high overhead expense of using operating rooms provided the impetus to develop more efficient nonsurgical oocyte retrieval techniques.
When was IVF invented?
In vitro fertilization, popularly referred to as IVF, has captured the attention of the public since its sensational introduction in 1978. Today assisted reproductive technology is available throughout most of the civilized world, and the practice is largely different from that used during the early days. Refinements in laboratory technology and clinical practice have allowed IVF to evolve into a medical procedure that is efficient, safe, readily accessible, and relatively affordable. More than 2 million IVF children have been born to date, and it is likely that continued enhancements will widen its appeal and applicability.
What is the procedure of transferring oocytes into the fallopian tube?
This procedure was known as zygote intrafallopian transfer (ZIFT) and it allowed the confirmation of fertilization but maintained some of the theoretical benefits of GIFT (Hamori et al 1988). However, the use of two laparoscopies, one for oocyte retrieval and the other for zygote transfer, was a major limitation of this approach.
When did ultrasound help with oocyte retrieval?
Improvements in ultrasonography during the 1980s catalyzed the evolution of modern outpatient oocyte retrieval. Using transabdominal ultrasound guidance, various methods of oocyte retrieval included percutaneous (Lenz and Lauritsen 1982), transvesical (Lenz et al 1981; Lenz and Lauritsen 1982), per-urethral (Parsons et al 1985), and transvaginal follicle aspiration (Dellenbach et al 1985). Further refinements in ultrasound transducers led to the use of transvaginal ultrasound guided transvaginal follicle aspiration (Figure 3). First reported in 1987, this oocyte retrieval technique quickly became the procedure of choice due to better visualization, finer control, and less patient discomfort compared with other available methods (Wikland et al 1987). Obviating the need for laparoscopy decreased the number of personnel, time and procedure expense, reduced the risks of surgery and general anesthesia, and provided greater patient acceptance. IVF cases transitioned from 1–2 hours of hospital-based operating room time to 10–15 minute procedures that can be performed in an office setting.
What is PCR testing for?
Following this, PCR-based tests amplifying DNA fragments with causative mutations specific for single gene disorders such as α-1 antitrypsin deficiency and cystic fibrosis allowed identification of unaffected embryos and resulted in normal children (Verlinsky et al 1990; Handyside et al 1992). Biopsies of polar bodies during meiosis rather than blastomeres during the cleavage stage were used in some of these cases. Although this approach may be less disruptive to the embryo, it only allowed analyses of maternal DNA and hence had limited clinical applications. In the last 15 years, the number of inherited disorders diagnosed at the preimplantation stage has expanded to over 40 diseases, and the advent of multiplex-PCR which simultaneously amplifies several DNA fragments in a single reaction greatly improved the accuracy of the analyses (Kuliev et al 1998).
How many embryos survived cryopreservation?
During the initial years of experimentation, at best approximately 50% of embryos survived the freeze/thaw process and resulted in a pregnancy rate of 13.4% per embryo transfer procedure, as only 4.6% of the individual thawed embryos implanted (Friedler et al 1988). By 2003, frozen embryo transfers accounted for 21 981 of the 112 872 IVF cycles (17.8%) performed in the US, with an overall live birth rate of 27.0% per embryo transfer procedure.
When did IVF start?
While advances in early IVF refined the technology for treating women with tubal disease, those with natural or premature ovarian failure had no effective fertility treatments until 1983. In December of that year, a 25 year old patient with secondary amenorrhea and premature ovarian failure became the first person to successfully deliver a pregnancy using a donor egg. Dr. Peter Renou of the Monash IVF group in Australia inseminated a single oocyte, donated by a 29-year-old patient undergoing IVF herself for tubal disease, with the sperm from the recipient’s husband. The embryo was transferred back into the uterus of the recipient and resulted in a healthy full term live-birth (Lutjen et al 1984).
How to evaluate hemocompatibility of biomaterials?
First, fresh human blood is collected and anticoagulated with low dose heparin . Thereafter, the test material is incubated at 37°C using static, agitated, or dynamic test models with the blood. The activation markers in the blood are analyzed before and after the incubation with the test material. Furthermore, the surface of the biomaterial is analyzed to determine the interaction of blood cells and proteins with the biomaterial surface.
How to measure erythrocytes?
The number of erythrocytes, leukocytes, and platelets is measured before and after the incubation of blood with biomaterial using a hematology analyzer, which uses electrical impedance . A decrease of the platelet count over time indicates a thrombogenic material. Furthermore, the rupture of erythrocytes, called hemolysis, is accompanied by the release of hemoglobin. Thus, an increased concentration of free hemoglobin in the plasma is a direct indicator of erythrocytes destruction. The damage of erythrocytes can lead to the reduced oxygen transport to tissues and organs in vivoand increased levels of free hemoglobin can induce toxicity or alter the kidney function (Qian et al., 2010). Additionally, microvesicles derived from erythrocytes can promote the thrombus formation in a tissue factor (TF)-dependent manner (Biró et al., 2003). Thus, hemolysis can be analyzed after direct or indirect blood contact. In direct analysis, blood is incubated with the biomaterial and in indirect testing blood is incubated with biomaterial extract (Kuhbier et al., 2017). Hemolysis can be detected by using a photometric colorimetric test (cyanmethemoglobin method) (Stadie, 1920). Thereby, the free amount of hemoglobin in plasma is examined after the addition of cyanmethemoglobin (CMH) reagent, e.g., Drabkin's reagent, which rapidly converts hemoglobin to the cyanoderivate (Neun and Dobrovolskaia, 2011). The absorption of CMH is measured at 540 nm using a photometer. Depending on hemolysis, materials can be classified in three different categories: Materials resulting in over 5% hemolysis are classified hemolytic, between 5 and 2% as slightly hemolytic, and below 2% as nonhemolytic (Totea et al., 2014).
Why is it important to collect blood?
The quality of collected blood is extremely important to enable standardized hemocompatibility analysis . The in vitroanalysis should be performed with fresh blood from healthy subjects (Blok et al., 2016). Blok et al. demonstrated that the stationary storage of blood over 4 h at room temperature affects the platelet function and activity of leukocytes. Thus, experiments should be started within 4 h after the blood collection. However, the faster the experiments are started, the better it is. Peripheral blood should be collected from healthy non-smoker, non-pregnant subjects free of medication (particularly drugs affecting the hemostasis, such as aspirin, oral contraceptives, and nonsteroidal anti-inflammatory drugs). Moreover, atraumatic blood collection by minimizing venostasis during blood withdrawal and the use of 21-gauge needles is required to minimize activation of platelets and the coagulation cascade during collection (Braune et al., 2013).
What is agitated flow model?
Static, agitated, or shear flow models are used to perform hemocompatibility studies. Before and after the incubation of the test material with fresh human blood, hemolysis, cell counts, and the activation of platelets, leukocytes, coagulation and complement system are analyzed.
When are activation markers analyzed?
The activation markers in the blood are analyzed before and after the incubation with the test material. Furthermore, the surface of the biomaterial is analyzed to determine the interaction of blood cells and proteins with the biomaterial surface. Incubation of biomaterials with human blood.
Which organ produces cytokines?
The endothelium produces cytokines, anti-thrombotic components and expresses adhesion molecules for thrombocytes, monocytes, and neutrophils and plays an important role in interaction between the circulating blood and injured vessel wall.
What are the different types of blood contact devices?
According this guideline, five different categories, thrombosis, coagulation, platelets, hematology, and immunology (complement system and leukocytes), are indicated for hemocompatibility evaluation. The devices are divided into three categories concerning blood contact: (1) Externally communicating devices with indirect blood contact, e.g., cannulas and blood collection sets; (2) Externally communicating devices with direct blood contact, e.g., catheters and hemodialysis equipment; (3) Implant devices , e.g., heart valves, stents, and vascular grafts. So far, several studies were performed according ISO-10993-4 to evaluate various blood contacting devices and materials, such as stents (Sinn et al., 2011; Stang et al., 2014), catheters with a noble metal alloy coating (Vafa Homann et al., 2016), poly(2-dimethylamino-ethylmethacrylate) (PDMAEMA) (Cerda-Cristerna et al., 2011), and DNA hydrogels (Stoll et al., 2017). To perform hemocompatibility analysis, static, agitated, or shear flow in vitromodels are used for the incubation of blood with the biomaterial. Before and after the incubation of biomaterials with fresh human blood, the activation markers regarding hemocompatibility are analyzed (Figure (Figure11).
How to detect dead cells in vitro?
Detection of dead cells is accomplished by measuring movement of molecules either into or out of cells across membranes that have become leaky and cannot be repaired. A major class of molecules that serve as indicators of dead cells include markers that exist in the cytoplasm of viable cells, but leak into the surrounding culture medium upon loss of membrane integrity. The marker can exist naturally such as an enzyme, or be introduced artificially, such as loading radioactive [51Cr] or a fluorescent marker into viable cells. Artificially introduced markers enable selective detection of target cell cytotoxicity for experiments using more than one population of cells such as cell mediated cytotoxicity. A second class of molecules that serves as an indicator of dead cells is referred to as “vital dyes”. These dyes typically are not permeable to viable cells, but can enter dead cells through damaged membranes. Examples include trypan blue and many fluorogenic DNA binding dyes. Addition of these molecules to cells in culture results in selective staining of the dead cells.
What is cytotoxicity assay?
Cytotoxicity Assays: In VitroMethods to Measure Dead Cells
How to tell if a cell is dead or alive?
Membrane integrity is the feature most often used to detect whether eukaryotic cells cultured in vitro are alive or dead. Cells that have lost membrane integrity and allow movement of otherwise non-permeable molecules are classified as non-viable or dead. Detection of dead cells is accomplished by measuring movement of molecules either into or out of cells across membranes that have become leaky and cannot be repaired. A major class of molecules that serve as indicators of dead cells include markers that exist in the cytoplasm of viable cells, but leak into the surrounding culture medium upon loss of membrane integrity. The marker can exist naturally such as an enzyme, or be introduced artificially, such as loading radioactive [51Cr] or a fluorescent marker into viable cells. Artificially introduced markers enable selective detection of target cell cytotoxicity for experiments using more than one population of cells such as cell mediated cytotoxicity. A second class of molecules that serves as an indicator of dead cells is referred to as “vital dyes”. These dyes typically are not permeable to viable cells, but can enter dead cells through damaged membranes. Examples include trypan blue and many fluorogenic DNA binding dyes. Addition of these molecules to cells in culture results in selective staining of the dead cells.
What is the purpose of artificial markers?
Artificially introduced markers enable selective detection of target cell cytotoxicity for experiments using more than one population of cells such as cell mediated cytotoxicity. A second class of molecules that serves as an indicator of dead cells is referred to as “vital dyes”.
Why do dyes cause cytotoxicity?
Dyes that cause cytotoxicity upon long term exposure to cells may be the result of partial permeability. Membrane permeability may depend on the cell type, the overall health of the cells or whether the dyes are substrates for efflux pumps that result in expulsion from the cytoplasm even if the dye does enter the cell.
How to determine if a cell is dead?
As illustrated in the diagram in Figure 1, assays used to detect dead cells include measuring the leakage of a component (usually an enzyme marker) from the cytoplasm into the culture medium or the penetration of an otherwise non-permeable dye into cells with a compromised membrane. This chapter will describe options for both general approaches for measuring dead cells.
What is the function of membrane integrity?
Membrane integrity is the feature most often used to detect whether eukaryotic cells cultured in vitro are alive or dead. Cells that have lost membrane integrity and allow movement of otherwise non-permeable molecules are classified as non-viable or dead. Detection of dead cells is accomplished by measuring movement of molecules ...
Why It's Done
- In vitro fertilization (IVF) is a treatment for infertility or genetic problems. If IVF is performed to treat infertility, you and your partner might be able to try less-invasive treatment options before attempting IVF, including fertility drugs to increase production of eggs or intrauterine inseminati…
Risks
- Risks of IVFinclude: 1. Multiple births. IVFincreases the risk of multiple births if more than one embryo is transferred to your uterus. A pregnancy with multiple fetuses carries a higher risk of early labor and low birth weight than pregnancy with a single fetus does. 2. Premature delivery and low birth weight. Research suggests that IVFslightly increases the risk that the baby will be …
How You Prepare
- The Centers for Disease Control and Prevention and the Society for Assisted Reproductive Technology provide information online about U.S. clinics' individual pregnancy and live birth rates. A clinic's success rate depends on many factors. These include patients' ages and medical issues, as well as the clinic's treatment population and treatment approaches. Ask for detailed informati…
What You Can Expect
- IVF involves several steps — ovarian stimulation, egg retrieval, sperm retrieval, fertilization and embryo transfer. One cycle of IVFcan take about two to three weeks. More than one cycle may be needed.
Results
- About 12 days to two weeks after egg retrieval, your doctor will test a sample of your blood to detect whether you're pregnant. 1. If you're pregnant,your doctor will refer you to an obstetrician or other pregnancy specialist for prenatal care. 2. If you're not pregnant, you'll stop taking progesterone and likely get your period within a week. If you don't get your period or you have un…
Clinical Trials
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