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This document appears to be the syllabus for a course titled "Advanced Animal Cell Culture" offered in the second semester of 2013 at Chungbuk National University's Department of Animal Science. The syllabus lists 8 topics that will be covered across 8 class dates from September to December. These topics include introductions to cell culture, using cell culture as a research model, producing antibodies and proteins through cell culture, protein production and purification techniques, and stem cell research. The document also provides details on assignments and presentations related to specific class dates.
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This document appears to be the syllabus for a 7th lecture course on Advanced Animal Cell Culture offered in the 2nd semester of 2013 at Chungbuk National University's Department of Animal Science. The syllabus lists 8 topics that will be covered across 8 class dates from September to December 2013. These topics include introductions to cell culture, using cell culture as a research model, antibody/protein production in cell culture, protein production and purification, stem cells, and generating transgenic/knockout animals and genome engineering techniques. It also lists which students will present on certain topics and dates.
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This document contains the syllabus for an advanced animal cell culture course offered in the second semester of 2013 at Chungbuk National University's Department of Animal Science. The syllabus lists 8 topics that will be covered across 8 classes between September and December, including introductions to cell culture, stem cells, transgenic animals, and genome engineering. It also provides the dates and instructors for the classes on cell culture for antibody and protein production, and protein production/purification.
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This document discusses cytotoxicity and methods for measuring it in vitro. It defines cytotoxicity as the ability of chemicals or cells to destroy living cells, which can lead to necrosis, apoptosis, or cytostasis. Measuring cytotoxicity is important for drug development and safety testing. In vitro assays are now commonly used as they are faster, cheaper, and more accurate than animal models. Several types of cytotoxicity assays are described, including dye exclusion assays like trypan blue, colorimetric assays like MTT, fluorometric assays like CFDA-AM, and luminometric assays like ATP assays. These assays measure endpoints such as membrane integrity, enzyme activity, proliferation, and ATP production to determine cytotoxic effects of chemicals on cells.
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Hybridoma technology is a method for generating monoclonal antibodies by fusing B cells with myeloma cells. Georges Köhler and César Milstein developed this technique in 1975 and were awarded the Nobel Prize for it. The hybridoma cells produced from this fusion have the antibody production ability of B cells and indefinite growth ability of myeloma cells, allowing large-scale monoclonal antibody production. Hybridomas are selected using HAT medium, which eliminates unfused B and myeloma cells. The resulting hybridomas are then screened and cultured to produce monoclonal antibodies, which have wide applications in areas like cancer diagnosis, treatment, and research.
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This document appears to be the syllabus for a 7th lecture course on Advanced Animal Cell Culture offered in the 2nd semester of 2013 at Chungbuk National University's Department of Animal Science. The syllabus lists 8 topics that will be covered across 8 class dates from September to December 2013. These topics include introductions to cell culture, using cell culture as a research model, antibody/protein production in cell culture, protein production and purification, stem cells, and generating transgenic/knockout animals and genome engineering techniques. It also lists which students will present on certain topics and dates.
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This document discusses cytotoxicity and methods for measuring it in vitro. It defines cytotoxicity as the ability of chemicals or cells to destroy living cells, which can lead to necrosis, apoptosis, or cytostasis. Measuring cytotoxicity is important for drug development and safety testing. In vitro assays are now commonly used as they are faster, cheaper, and more accurate than animal models. Several types of cytotoxicity assays are described, including dye exclusion assays like trypan blue, colorimetric assays like MTT, fluorometric assays like CFDA-AM, and luminometric assays like ATP assays. These assays measure endpoints such as membrane integrity, enzyme activity, proliferation, and ATP production to determine cytotoxic effects of chemicals on cells.
05 Monoclonal Antibodies
Monoclonal antibodies are produced through the fusion of antibody-producing B cells with myeloma cells to form immortal hybridoma cells. This process allows for the mass production of antibodies that are identical and specifically target a single antigen or macromolecule. Monoclonal antibodies have important applications in disease diagnosis, treatment, and pregnancy testing due to their high specificity and ability to identify pathogens, tumors, and other targets.
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Hybridoma technology is a method for generating monoclonal antibodies by fusing B cells with myeloma cells. Georges Köhler and César Milstein developed this technique in 1975 and were awarded the Nobel Prize for it. The hybridoma cells produced from this fusion have the antibody production ability of B cells and indefinite growth ability of myeloma cells, allowing large-scale monoclonal antibody production. Hybridomas are selected using HAT medium, which eliminates unfused B and myeloma cells. The resulting hybridomas are then screened and cultured to produce monoclonal antibodies, which have wide applications in areas like cancer diagnosis, treatment, and research.
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3. Various methods are used to monitor and determine cell growth in culture, including counting cells with a hemocytometer, measuring protein, DNA, or glucose levels, or assessing metabolic activity or viability.
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2) Chromatography techniques separate proteins based on properties like binding interactions, charge, size, and isoelectric point using columns.
3) The final purity required depends on the application, with 95-99% purity needed for assays and over 99% for therapeutic use. Analytical techniques confirm identity and purity of the purified protein.
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Chromatography is a technique used to separate mixtures by exploiting differences in how components interact with two phases - a stationary phase and a mobile phase. There are various types including thin layer chromatography, column chromatography, gas chromatography, and high performance liquid chromatography. Each type uses different stationary and mobile phases to separate components based on properties like size, charge, or binding affinity. Chromatography is widely used in science and industry for analytical and preparative purposes such as purifying compounds.
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Humanized antibody
Humanized antibodies are antibodies made from non-human species that have been modified to increase their similarity to human antibodies. They have progressed from murine antibodies to chimeric to humanized to fully human antibodies. Humanized antibodies can be prepared using transgenic mice or phage display technologies. Phage display involves inserting antibody genes into phages and selecting desirable proteins by screening against antigens. The selected proteins can then be produced and assembled into functional antibodies.
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This document discusses primary cells and their use in biomedical research. Primary cells are isolated directly from tissues and have important advantages over cell lines, including greater biological relevance. Primary cells are used in applications such as biomarker research, drug development, immunotherapeutics, and tissue engineering. The document reviews techniques for working with primary cells, including isolation, culture, differentiation, and characterization using methods like flow cytometry and immunoassays. Primary cells provide a more physiologically relevant model for research compared to cell lines.
Cell cell hybridization or somatic cell hybridization
What is Cell-Cell Hybridization?
History
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Mapping of genes by somatic cell Hybridization
Hybridoma technology
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Somatic cell hybridization
Somatic cell hybridization involves fusing cells from two different species, such as human and mouse cells, to form hybrid cells containing chromosomes from both species. This technique allows genes to be mapped to specific chromosomes. It works by using selective growth conditions that require the hybrid cell to retain certain human chromosomes in order to survive. Over successive cell divisions, human chromosomes are eliminated at random except for those required for survival. This allows the creation of cell lines containing partial sets of human chromosomes that can be analyzed to correlate genes with specific chromosomes. The technique has been important for mapping the human genome.
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- Cell culture techniques have progressed significantly since the late 1800s, becoming a widely accepted research tool by the 1980s.
- Cells cultured in vitro can be primary cultures directly from tissue, or secondary/continuous cultures derived from primary cultures that are serially passaged. Continuous cultures are immortalized, having an infinite lifespan.
- Cells require specific culture conditions including nutrients, temperature, pH, humidity and sterility to grow. Basal media provides nutrients and supplements like serum provide growth factors.
- Routine passaging maintains cell viability by transferring to fresh media and surfaces. Cryopreservation allows long-term storage in liquid nitrogen. Contamination control is important for consistent results.
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Single Cell Isolation
Single cell analysis is getting into the focus of many research fields as it demonstrates the individual contribution of every cell in a heterogeneous population without obscuring a biological response that may otherwise occur when cells are assessed in bulk. Although single-cell research is currently gaining momentum, yet it is challenged by the lack of affordable methods to precisely isolate a single cell from a heterogeneous cell population without causing high cellular stress.
The QIAscout system is an effective and fast method to isolate viable single cells ensuring minimal manipulation of the cellular status. The QIAscout array is ideal for various cell types like adherent cells, suspension cells, cell lines, primary cells, and fluorescent cell lines providing an environment suitable for their growth and viability similar to any standard cell culture dish. The QIAscout array consists of 12,000 microrafts that can be selectively isolated containing the single cell of interest.
This novel single cell isolation method is ideal to separate single cells for further downstream analysis or cultivation of clonal sub-populations. Single cell isolation with QIAscout is compatible with multiple downstream applications such as whole genome and transcriptome amplification methods, PCR and NGS.
Somatic hybridization faizy
This document provides an introduction to somatic hybridization, which is the fusion of somatic protoplasts from two different plant species or varieties to create hybrid plants. It discusses the history and development of the technique, including early work isolating protoplasts. The key steps in somatic hybridization are described: isolating protoplasts using either mechanical or enzymatic methods, fusing the protoplasts spontaneously or through induction methods, identifying and selecting hybrid cells, culturing the hybrid cells, and regenerating hybrid plants. Advantages include creating novel hybrids and transferring desirable traits between species, while limitations include low regeneration rates and viability of fused products.
Cell proliferation assay services
Cell proliferation is the process by which the number of cells increases through cell division and differentiation. It plays a key role in development and tissue homeostasis. There are several techniques to measure cell proliferation, including assays that detect proliferation markers, measure ATP or new RNA synthesis, track membrane integrity, or analyze DNA or metabolic activity. Creative Bioarray offers flexible and accurate cell proliferation assays using various techniques such as DNA synthesis assays with thymidine or BrdU labeling or metabolic assays measuring tetrazolium salts or Alamar Blue, which provide dose response curves and IC50 values with a fast turnaround time.
Monoclonal antibodies
Monoclonal antibodies are antibodies produced by a single clone of cells that make one specific antibody. They are homogeneous, specific, and do not vary between batches as they bind to a single antigen. Monoclonal antibodies have applications in diagnostics, such as pregnancy tests, and therapeutics, such as treatments for cancer and organ transplantation. They are produced through cell fusion methods like roller bottle culture or bioreactor culture.
Somatic hybridization
Somatic hybridization involves fusing isolated plant protoplasts to combine their genomes. This allows crossing of species, genera, or even kingdoms that cannot naturally hybridize. Protoplasts lack cell walls, making them easy to fuse in vitro. Fusion can be spontaneous or induced using chemicals, mechanical means, or electricity. Induced fusion allows crossing of incompatible plants. Resulting plants or cells containing genomes from both parents are called somatic hybrids or cybrids. Somatic hybridization has applications in breeding new inter-specific hybrids, restoring fertility, and transferring genes for traits. However, low regeneration rates and hybrid viability limit its effectiveness.
12. Cell synchronization and Immortalization
Cell synchronization helps in obtaining distinct sub population of cells representing different stages of cell cycle.It helps in collecting population wide data of cells progressing through various stages of cell cycle. Immortalization, refers to cells having capability of undergoing cell division infinitely. Immortal cells are particularly preferred in cell culture to enable long time storage and use. This presentation teaches about cell synchronization, methods of cell synchronization, cellular transformation, immortalization and mechanism of immortalization.
Lecture 6 cell culture monitoring
1. Cell banks are used to store and maintain animal cell lines, with a master cell bank storing early passage cells and a working cell bank storing cells from the master bank for several passages.
2. Cells are cryopreserved in liquid nitrogen by slow freezing and fast thawing to maintain viability, with cryoprotectants like glycerol or DMSO added to minimize ice crystal formation.
3. Various methods are used to monitor and determine cell growth in culture, including counting cells with a hemocytometer, measuring protein, DNA, or glucose levels, or assessing metabolic activity or viability.
Monoclonal antibodies
Monoclonal antibodies are produced through the hybridoma technology developed by Kohler and Milstein in 1975. This involves fusing B cells from an immunized animal with myeloma cells to produce hybridomas that secrete a single antibody specific to the target antigen. The antibodies can be screened and a clone selected to mass produce monoclonal antibodies that recognize a single epitope. Monoclonal antibodies have various applications including disease diagnosis, immunotherapy, and immunosuppression for organ transplants. While powerful tools, limitations include potential immunogenicity and high production costs.
Arsh copy
Cell lines are immortal cell cultures that are used extensively in biomedical research. Some key uses of cell lines include studying basic cell biology, testing drug toxicity, cancer research, virology studies, and gene therapy. Common cell lines include HeLa cells from cervical cancer tissue and MCF-7 cells from breast cancer tissue. Cell lines are also important in the production of vaccines, antibodies, and other biological products through recombinant protein expression in cultured cells. Respiratory and pancreatic beta cell lines have applications in research on inflammation, diabetes, and other diseases.
Cell lines Basics
This work is done in IIT-M (Indian Institute of Technology- Madras) with help of Indian Academy of Science during June 2011-Oct 2011 under Dr Karunagaran Devarajan sir
Monoclonal Antibody
The document presents information on monoclonal antibodies. It discusses that monoclonal antibodies are identical antibodies produced through cell fusion techniques from a single parent cell. The summary includes that monoclonal antibodies have characteristics of homogeneity and specificity. The document also outlines the structure, advantages, disadvantages, production methods and applications of monoclonal antibodies such as in cancer treatment, immunosuppression, and autoimmune diseases. It concludes that monoclonal antibodies offer unique target specificity and can be engineered to reduce immunogenicity issues.
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This document discusses synchronization of suspension cell cultures, cellular totipotency, and cytodifferentiation. Synchronization aims to make cells progress through the cell cycle simultaneously and can be achieved through physical methods like temperature shocks or chemical methods like starvation or using inhibitors. Cellular totipotency refers to a cell's ability to produce all cell types and organize into a whole organism. Cytodifferentiation is the process by which dedifferentiated callus cells undergo quiescence and senescence leading to differentiation of vascular tissues during growth and maturation.
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3) The final purity required depends on the application, with 95-99% purity needed for assays and over 99% for therapeutic use. Analytical techniques confirm identity and purity of the purified protein.
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みなさんこんにちはこれ何文字まで入るの？40文字以下不可とか本当に意味わからないけどこれ限界文字数書いてないからマジでやばい文字数いけるんじゃないの？えこ...
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Cell culture 04
- 1. 4rd Lecture Advanced Aminal Cell Culture 2013 2nd Semester Department of Animal Science Chungbuk National University
- 2. Syllabus Date Topics September 5, 2013 Introduction : What is Cell Culture? September 12, 2013 Cell Culture As Model System For Research September 26, 2013 Cell Culture For Antibody / Protein Production October 17, 2013 Protein Production/Purification October 31, 2013 Stem Cell I November 14, 2013 Stem Cell II November 28, 2013 TG/KO Animals December 5, 2013 Genome Engineering/NGS December 12, 2013 Final Exam
- 3. Date September 26, 2013 Cell Culture For Antibody / Protein Production 조유미, Madhusumida October 17, 2013 Protein Production/Purification 이미진, 정용호 이영, 윤준호 October 31, 2013 November 14, 2013 Stem Cell I Jia Jia Lin, 염동현 November 28, 2013 Stem Cell II Zhao MingHui,권정우 December 5, 2013 Transgenic Animals Lin Zili, 이상배 December 12, 2013 Genome Engineering/NGS 조유진, 김상욱
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- 5. Protein Purification Principles - Ion-exchnage Different protein has different ionic properites (Positive or Negative) - Gel Filtration Seperation based on Protein Size - Affinity Some protein bind specific ligands - UltraFiltration Concentration of Protein / Desalting
- 6. Ion-exchange chromatography - Purification based on protein surface charge
- 7. - Anion Exchange Chromatography Resin has (+) charge on it Negative (anion) charged protein bound on the resin By increase of anion (Cl-) concetration, protein eluted from the resin Q-Sepharose : Strong Anion Exchanger DEAE-Sephasrose : Moderately strong anion exchanger
- 8. - Cation Exchange Chromatography Resin has (-) charge on it Positive (cation charged protein bound on the resin By increase of cation(For example, Na+) concentration, protein eluted from the resin S-Sepharose : Strong cation Exchanger CM-Sepharose : Moderately strong cation exchanger
- 9. Examples of Ion-exchange chromatography resin Mono-Q and Mono-S : Strong anion (Q) and cation (S) exchanger
- 10. General Procedure of Ion Exchange Chromatography 1. Load Sample Unbound protein goes to flow through column 2. Wash Sample with low salt buffer 3. Increase Salt Concentration by Gradients Lower Salt : Weak binding Higher Salt : Strong binding
- 11. Pros It can bind very large amount of sample : Ideal for initial capturing of desired protein Good for the sample concentration If it is optimized, it can increase purification yield significantly General Purpose chromatography (You can use it most of soluble proteins) Cons Sample should be stable at low salt (less than 100mM) buffer Procedure should be optimized (case by case)
- 12. Affinity Chromatography - Use resin which can bind specifically desired protein - Examples : Protein A sepharose : bind to IgG GSH sepharose : Glutathione transferase Ni-NTA Sepharose : PolyHistidine Chitin bead : Cellulose binding protein - Before genetic manipulation, Affinity chromatography was very limited usages. - But now it is one of major techniques for protein purifications
- 13. Affinity tag addition using recombinant RNA technology - Most of protein does not have specific affinity bead to bind. - We can add specific tag on protein to facilliate purification Target Protein Gene Affinity tag Gene Affinity Tag Affinity Bead Affinity Tag Target Protein Gene Target Protein Gene
- 14. PolyHistidine Tag (His-tag) : Can bind to Ni-NTA bead Blue Color From Nickel Glutathione S-transferase Tag (GST-tag) : Can bind to Glutathione Sepharose FLAGTM tag (DYKDDDDK) : Can bind Flag-tag recognize antibody
- 16. • Pros - Very high level of purification can be achieved - Initial Capturing • - Cons Limited Affinity Resin At least modification of protein is required Cleavage of Tag
- 17. Size Exclusion Chromatography - Separate protein based on sizes - Small protein enter bead, while big protein migrate fast - Mainly used for the final step of purification (Protein Polishing)
- 19. • Pros - Can separate protein based on the oligomeric state - Final Polishing of protein • - Cons Seperation depend on the loading volumes Sample should be highly concentrated Limitation of protein amount
- 20. Multistep purification Affinity chromatography (Initial capturing) Ion exchange chromatography (Intermediate purification) Size Exclusionchromatography (Polishing Step)
- 21. Measurement of Purity SDS-PAGE Endotoxin detection -Endotoxin : Generally refer ‘bacterial originated lipoplysaccharide’ (LPS) - Induces unwanted immune response - Should be removed for the biologic productions