This document discusses animal tissue culture and cell culture techniques. It begins by defining tissue culture as the removal and growth of cells, tissues or organs from animals or plants in an artificial environment that supplies nutrients for growth. It then covers major developments in the field like the use of antibiotics, trypsin for subculturing, and chemically defined media. Applications of cell culture discussed include research areas like toxicology testing, cancer research, virology and genetic engineering. The document also covers primary culture, cell lines, monolayer and suspension culture systems, culturing adherent and suspension cells, cryopreservation of cells, cell viability assessment, and basic cell culture equipment.
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance a...Ananya Sinha
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance and application
Generally, suspension culture is a one stop technology to produce secondary metabolites on a large scale in-vitro, irrespective of the climatic condition or nutrient availability (as required in field plants).
In this presentation, we will see the importance of suspension culture, culturing methods and it's application (mostly with respect to plants) and also focus on what exactly is a single cell culture.
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Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
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2 Case Reports of Gastric Ultrasound
Acute scrotum is a general term referring to an emergency condition affecting the contents or the wall of the scrotum.
There are a number of conditions that present acutely, predominantly with pain and/or swelling
A careful and detailed history and examination, and in some cases, investigations allow differentiation between these diagnoses. A prompt diagnosis is essential as the patient may require urgent surgical intervention
Testicular torsion refers to twisting of the spermatic cord, causing ischaemia of the testicle.
Testicular torsion results from inadequate fixation of the testis to the tunica vaginalis producing ischemia from reduced arterial inflow and venous outflow obstruction.
The prevalence of testicular torsion in adult patients hospitalized with acute scrotal pain is approximately 25 to 50 percent
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
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Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
1. Animal Tissue Culture
PRESENTED BY
ASHUTOSH B. MAHALE
M.PHARMACY FIRST YEAR ( PHARMACOLOGY )
UNIVERSITY DEPARTMENT OF PHARMACEUTICAL SCIENCES, R.T.M NAGPUR
UNIVERSITY , NAGPUR
22/11/17 1
2. Introduction
Cell culture has become one of the major tools used in
the life sciences today.
Tissue Culture is the general term for the removal
of cells, tissues, or organs from an animal
or plant and their subsequent placement
into an artificial environment
conducive to growth. This
environment usually consists
of a suitable glass or plastic
culture vessel containing a
liquid or semisolid medium
that supplies the nutrients
essential for survival & growth.
22/11/17 2
3. Major development’s in cell culture
technology
• First development was the use of antibiotics
which inhibits the growth of contaminants.
• Second was the use of trypsin to remove
adherent cells to subculture further from the
culture vessel
• Third was the use of chemically defined
culture medium.
22/11/17 3
4. Why is cell culture used for?
Areas where cell culture technology is currently
playing a major role.
• Model systems for
Studying basic cell biology, interactions between disease
causing agents and cells, effects of drugs on cells, process and
triggering of aging & nutritional studies.
• Toxicity testing
Study the effects of new drugs.
• Cancer research
Study the function of various chemicals, virus &
radiation to convert normal cultured cells to cancerous cells.
22/11/17 4
5. • Virology - Cultivation of virus for vaccine production, also
used to study there infectious cycle.
• Genetic Engineering
Production of commercial proteins, large scale
production of viruses for use in vaccine production e.g.
polio, rabies, chicken pox, hepatitis B & measles
• Gene therapy
Cells having a functional gene can be replaced to
cells which are having non-functional gene
22/11/17 5
6. Tissue culture
• In vitro cultivation of organs, tissues & cells at defined
temperature using an incubator & supplemented with a medium
containing cell nutrients & growth factors is collectively known
as tissue culture
• Different types of cell grown in culture includes connective
tissue elements such as fibroblasts, skeletal tissue, cardiac,
epithelial tissue (liver, breast, skin, kidney) and many different
types of tumor cells.
22/11/17 6
7. Primary culture
• Cells when surgically or enzymatically removed from an organism
and placed in suitable culture environment will attach and grow
are called as primary culture.
• Primary cells have a finite life span.
• Primary culture contains a very heterogeneous population of cells.
• Sub culturing of primary cells leads to the generation of cell lines.
• Cell lines have limited life span, they passage several times before
they become senescent.
• Lineage of cells originating from the primary culture is called a
cell strain.
22/11/17 7
8. Cell Culture Systems
• Two basic culture systems are used for growing
cells. These are based primarily upon the ability of
the cells to either grow attached to a glass or
treated plastic substrate (Monolayer Culture
Systems) or floating free in the culture medium
(Suspension Culture Systems).
22/11/17 8
9. Types of cells
On the basis of morphology (shape & appearance) or on
their functional characteristics. They are divided into three.
Epithelial like-attached to a substrate and appears
flattened and polygonal in shape
Lymphoblast like- cells do not attach remain in
suspension with a spherical shape
Fibroblast like- cells attached to an substrate appears
elongated and bipolar
22/11/17 9
10. Why sub culturing.?
• Once the available substrate surface is covered by
cells growth slows & ceases.
• Cells to be kept in healthy & in growing state have
to be sub-cultured or passaged.
• It’s the passage of cells when they reach to 80-
90% confluency in flask/dishes/plates.
• Enzyme such as trypsin, dipase, collagenase in
combination with EDTA breaks the cellular glue
that attached the cells to the surface.22/11/17 10
11. Culturing of cells
• Cells are cultured as anchorage dependent or independent
• Cell lines derived from normal tissues are considered as
anchorage-dependent grows only on a suitable substrate
e.g. tissue cells
• Suspension cells are anchorage
-independent e.g. blood cells
• Transformed cell lines either grows
as monolayer or as suspension
22/11/17 11
12. Adherent cells
• Cells which are anchorage dependent
• Cells are washed with PBS (free of Ca & Mg ) solution.
•
•
•
•
•
•
Add enough trypsin/EDTA to cover the monolayer
Incubate the plate at 37o C for 1-2 mts
Tap the vessel from the sides to dislodge the cells
Add complete medium to dissociate and dislodge the cells
with the help of pipette which are remained to be adherent
Add complete medium depends on the subculture
requirement either to 75 cm or 175 cm flask
22/11/17 12
13. Suspension cells
• Easier to passage as no need to detach them.
• As the suspension cells reach to confluency, asceptically
remove 1/3rd of medium replaced with the same amount of
pre-warmed medium.
22/11/17 13
14. • Vial from liquid nitrogen is placed into 370 C water bath, agitate
vial continuously until medium is thawed.
• Centrifuge the vial for 10 min. at 1000 rpm at RT, wipe top of vial
with 70% ethanol and discard the supernatant
• Resuspend cell pellet in 1 ml of complete medium with 20% PBS &
transfer to properly labeled culture plate
containing appropriate amount of medium.
• Check the cultures after 24 hrs to
ensure that they are attached to the plate
• Change medium as colour changes, use
20% PBS until the cells are established
Working with cryopreserved
cells
22/11/17 14
15. Freezing cells for storage
• Remove the growth medium, wash the cells by PBS and remove the
PBS by aspiration
• Dislodge the cells by trypsin-versene
• Dilute the cells with growth medium
• Transfer the cell suspension to a 15 ml conical tube, centrifuge at
200g for 5 mts at RT and remove the growth medium by aspiration
• Resuspend the cells in 1-2ml of freezing medium
• Transfer the cells to cryovials, incubate the cryovials at -80o C
overnight
• Next day transfer the cryovials to Liquid nitrogen22/11/17 15
16. Cell viability
• Cell viability is determined by staining the cells
with trypan blue.
• As trypan blue dye is permeable to non-viable cells
or dead cells whereas it is impermeable to this dye
• Stain the cells with trypan dye and load to
haemocytometer and calculate % of viable cells
% of viable
cells =
No. of unstained
cells x 100
22/11/17 16
17. Basic equipments used in cell culture
• Laminar cabinet-Vertical are preferable.•
Incubation facilities- Temperature of 25-300 C for insect & 370 C for
mammalian cells, CO2 2-5% & 95% air at 99% relative humidity.• Refrigerators- Liquid media kept at 40 C, enzymes (e.g. trypsin)
• Microscope An inverted microscope with 10x -100x.
• Tissue culture ware- Culture plastic ware of polystyrene.
22/11/17 17