Cell culture involves growing cells under controlled conditions outside of their natural environment. There are several types of culture, including organ culture, tissue culture, and cell culture. Cell culture allows cells to be studied without the complexity of an entire organism and provides a model for understanding cell behavior. Key factors that must be controlled include the culture surface, growth media formulation, temperature, gas exchange, and passaging of cells to maintain healthy growth.
Animal Cell Culture: Growth of animal cells in culture. PHARMACEUTICAL MICROB...Ms. Pooja Bhandare
PHARMACEUTICAL MICROBIOLOGY (BP303T)Unit-VPart-4
Animal Cell Culture: Growth of animal cells in culture.
Introduction: Histroy, The culture media used for animal cell culture are classified as,
Natural, Artificial, Synthesized
Natural Culture Media:
a. Blood Plasma:
b. Blood Serum:
c. Tissue Extracts:
Artificial Media
Some common examples of artificial media are,
Minimal Essential Medium (MEM),
CMRL 1066,
RPMI 1640.
Synthetic media re classified as,
Serum Containing Media.
Serum Free Media.
a. Serum Containing Media:
b. Serum Free Media:
Physicochemical Parameters needed for growth animal cell culture:
General procedure for cell Culture.
Isolation of the tissue:
Disaggregation of the Tissue:
Mechanical disaggregation
b. Enzymatic Disaggregation
. Trypsin based disaggregation or trypsinization:
Warm trypsinization:
Cold trypsinization:
Drawbacks of trypsin disaggregation:
B. Collagenase based disaggregation:
C. Chelating Agents:
3. Seeding of Culture:
Introduction
Terminologies
Types of tissue culture
Applications
Culturing
Sub-culturing
Cryopreservation
Detection of contaminants
In vitro transformation of cells
Cell viability
Rules for working in the Lab
Advantages
Limitations
Applications of fish celllines by B.pptxB. BHASKAR
Recent research studies on fish cell lines found many more application of cell lines pathological studies, toxicology, biomedical research, vaccine development etc
Animal Cell Culture: Growth of animal cells in culture. PHARMACEUTICAL MICROB...Ms. Pooja Bhandare
PHARMACEUTICAL MICROBIOLOGY (BP303T)Unit-VPart-4
Animal Cell Culture: Growth of animal cells in culture.
Introduction: Histroy, The culture media used for animal cell culture are classified as,
Natural, Artificial, Synthesized
Natural Culture Media:
a. Blood Plasma:
b. Blood Serum:
c. Tissue Extracts:
Artificial Media
Some common examples of artificial media are,
Minimal Essential Medium (MEM),
CMRL 1066,
RPMI 1640.
Synthetic media re classified as,
Serum Containing Media.
Serum Free Media.
a. Serum Containing Media:
b. Serum Free Media:
Physicochemical Parameters needed for growth animal cell culture:
General procedure for cell Culture.
Isolation of the tissue:
Disaggregation of the Tissue:
Mechanical disaggregation
b. Enzymatic Disaggregation
. Trypsin based disaggregation or trypsinization:
Warm trypsinization:
Cold trypsinization:
Drawbacks of trypsin disaggregation:
B. Collagenase based disaggregation:
C. Chelating Agents:
3. Seeding of Culture:
Introduction
Terminologies
Types of tissue culture
Applications
Culturing
Sub-culturing
Cryopreservation
Detection of contaminants
In vitro transformation of cells
Cell viability
Rules for working in the Lab
Advantages
Limitations
Applications of fish celllines by B.pptxB. BHASKAR
Recent research studies on fish cell lines found many more application of cell lines pathological studies, toxicology, biomedical research, vaccine development etc
QA Paediatric dentistry department, Hospital Melaka 2020Azreen Aj
QA study - To improve the 6th monthly recall rate post-comprehensive dental treatment under general anaesthesia in paediatric dentistry department, Hospital Melaka
Struggling with intense fears that disrupt your life? At Renew Life Hypnosis, we offer specialized hypnosis to overcome fear. Phobias are exaggerated fears, often stemming from past traumas or learned behaviors. Hypnotherapy addresses these deep-seated fears by accessing the subconscious mind, helping you change your reactions to phobic triggers. Our expert therapists guide you into a state of deep relaxation, allowing you to transform your responses and reduce anxiety. Experience increased confidence and freedom from phobias with our personalized approach. Ready to live a fear-free life? Visit us at Renew Life Hypnosis..
India Clinical Trials Market: Industry Size and Growth Trends [2030] Analyzed...Kumar Satyam
According to TechSci Research report, "India Clinical Trials Market- By Region, Competition, Forecast & Opportunities, 2030F," the India Clinical Trials Market was valued at USD 2.05 billion in 2024 and is projected to grow at a compound annual growth rate (CAGR) of 8.64% through 2030. The market is driven by a variety of factors, making India an attractive destination for pharmaceutical companies and researchers. India's vast and diverse patient population, cost-effective operational environment, and a large pool of skilled medical professionals contribute significantly to the market's growth. Additionally, increasing government support in streamlining regulations and the growing prevalence of lifestyle diseases further propel the clinical trials market.
Growing Prevalence of Lifestyle Diseases
The rising incidence of lifestyle diseases such as diabetes, cardiovascular diseases, and cancer is a major trend driving the clinical trials market in India. These conditions necessitate the development and testing of new treatment methods, creating a robust demand for clinical trials. The increasing burden of these diseases highlights the need for innovative therapies and underscores the importance of India as a key player in global clinical research.
CHAPTER 1 SEMESTER V PREVENTIVE-PEDIATRICS.pdfSachin Sharma
This content provides an overview of preventive pediatrics. It defines preventive pediatrics as preventing disease and promoting children's physical, mental, and social well-being to achieve positive health. It discusses antenatal, postnatal, and social preventive pediatrics. It also covers various child health programs like immunization, breastfeeding, ICDS, and the roles of organizations like WHO, UNICEF, and nurses in preventive pediatrics.
Defecation
Normal defecation begins with movement in the left colon, moving stool toward the anus. When stool reaches the rectum, the distention causes relaxation of the internal sphincter and an awareness of the need to defecate. At the time of defecation, the external sphincter relaxes, and abdominal muscles contract, increasing intrarectal pressure and forcing the stool out
The Valsalva maneuver exerts pressure to expel faeces through a voluntary contraction of the abdominal muscles while maintaining forced expiration against a closed airway. Patients with cardiovascular disease, glaucoma, increased intracranial pressure, or a new surgical wound are at greater risk for cardiac dysrhythmias and elevated blood pressure with the Valsalva maneuver and need to avoid straining to pass the stool.
Normal defecation is painless, resulting in passage of soft, formed stool
CONSTIPATION
Constipation is a symptom, not a disease. Improper diet, reduced fluid intake, lack of exercise, and certain medications can cause constipation. For example, patients receiving opiates for pain after surgery often require a stool softener or laxative to prevent constipation. The signs of constipation include infrequent bowel movements (less than every 3 days), difficulty passing stools, excessive straining, inability to defecate at will, and hard feaces
IMPACTION
Fecal impaction results from unrelieved constipation. It is a collection of hardened feces wedged in the rectum that a person cannot expel. In cases of severe impaction the mass extends up into the sigmoid colon.
DIARRHEA
Diarrhea is an increase in the number of stools and the passage of liquid, unformed feces. It is associated with disorders affecting digestion, absorption, and secretion in the GI tract. Intestinal contents pass through the small and large intestine too quickly to allow for the usual absorption of fluid and nutrients. Irritation within the colon results in increased mucus secretion. As a result, feces become watery, and the patient is unable to control the urge to defecate. Normally an anal bag is safe and effective in long-term treatment of patients with fecal incontinence at home, in hospice, or in the hospital. Fecal incontinence is expensive and a potentially dangerous condition in terms of contamination and risk of skin ulceration
HEMORRHOIDS
Hemorrhoids are dilated, engorged veins in the lining of the rectum. They are either external or internal.
FLATULENCE
As gas accumulates in the lumen of the intestines, the bowel wall stretches and distends (flatulence). It is a common cause of abdominal fullness, pain, and cramping. Normally intestinal gas escapes through the mouth (belching) or the anus (passing of flatus)
FECAL INCONTINENCE
Fecal incontinence is the inability to control passage of feces and gas from the anus. Incontinence harms a patient’s body image
PREPARATION AND GIVING OF LAXATIVESACCORDING TO POTTER AND PERRY,
An enema is the instillation of a solution into the rectum and sig
Global launch of the Healthy Ageing and Prevention Index 2nd wave – alongside...ILC- UK
The Healthy Ageing and Prevention Index is an online tool created by ILC that ranks countries on six metrics including, life span, health span, work span, income, environmental performance, and happiness. The Index helps us understand how well countries have adapted to longevity and inform decision makers on what must be done to maximise the economic benefits that comes with living well for longer.
Alongside the 77th World Health Assembly in Geneva on 28 May 2024, we launched the second version of our Index, allowing us to track progress and give new insights into what needs to be done to keep populations healthier for longer.
The speakers included:
Professor Orazio Schillaci, Minister of Health, Italy
Dr Hans Groth, Chairman of the Board, World Demographic & Ageing Forum
Professor Ilona Kickbusch, Founder and Chair, Global Health Centre, Geneva Graduate Institute and co-chair, World Health Summit Council
Dr Natasha Azzopardi Muscat, Director, Country Health Policies and Systems Division, World Health Organisation EURO
Dr Marta Lomazzi, Executive Manager, World Federation of Public Health Associations
Dr Shyam Bishen, Head, Centre for Health and Healthcare and Member of the Executive Committee, World Economic Forum
Dr Karin Tegmark Wisell, Director General, Public Health Agency of Sweden
R3 Stem Cells and Kidney Repair A New Horizon in Nephrology.pptxR3 Stem Cell
R3 Stem Cells and Kidney Repair: A New Horizon in Nephrology" explores groundbreaking advancements in the use of R3 stem cells for kidney disease treatment. This insightful piece delves into the potential of these cells to regenerate damaged kidney tissue, offering new hope for patients and reshaping the future of nephrology.
Medical Technology Tackles New Health Care Demand - Research Report - March 2...pchutichetpong
M Capital Group (“MCG”) predicts that with, against, despite, and even without the global pandemic, the medical technology (MedTech) industry shows signs of continuous healthy growth, driven by smaller, faster, and cheaper devices, growing demand for home-based applications, technological innovation, strategic acquisitions, investments, and SPAC listings. MCG predicts that this should reflects itself in annual growth of over 6%, well beyond 2028.
According to Chris Mouchabhani, Managing Partner at M Capital Group, “Despite all economic scenarios that one may consider, beyond overall economic shocks, medical technology should remain one of the most promising and robust sectors over the short to medium term and well beyond 2028.”
There is a movement towards home-based care for the elderly, next generation scanning and MRI devices, wearable technology, artificial intelligence incorporation, and online connectivity. Experts also see a focus on predictive, preventive, personalized, participatory, and precision medicine, with rising levels of integration of home care and technological innovation.
The average cost of treatment has been rising across the board, creating additional financial burdens to governments, healthcare providers and insurance companies. According to MCG, cost-per-inpatient-stay in the United States alone rose on average annually by over 13% between 2014 to 2021, leading MedTech to focus research efforts on optimized medical equipment at lower price points, whilst emphasizing portability and ease of use. Namely, 46% of the 1,008 medical technology companies in the 2021 MedTech Innovator (“MTI”) database are focusing on prevention, wellness, detection, or diagnosis, signaling a clear push for preventive care to also tackle costs.
In addition, there has also been a lasting impact on consumer and medical demand for home care, supported by the pandemic. Lockdowns, closure of care facilities, and healthcare systems subjected to capacity pressure, accelerated demand away from traditional inpatient care. Now, outpatient care solutions are driving industry production, with nearly 70% of recent diagnostics start-up companies producing products in areas such as ambulatory clinics, at-home care, and self-administered diagnostics.
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Antibiotic Stewardship by Anushri Srivastava.pptxAnushriSrivastav
Stewardship is the act of taking good care of something.
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS) in 2015 to fill knowledge gaps and inform strategies at all levels.
ACCORDING TO apic.org,
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
ACCORDING TO pewtrusts.org,
Antibiotic stewardship refers to efforts in doctors’ offices, hospitals, long term care facilities, and other health care settings to ensure that antibiotics are used only when necessary and appropriate
According to WHO,
Antimicrobial stewardship is a systematic approach to educate and support health care professionals to follow evidence-based guidelines for prescribing and administering antimicrobials
In 1996, John McGowan and Dale Gerding first applied the term antimicrobial stewardship, where they suggested a causal association between antimicrobial agent use and resistance. They also focused on the urgency of large-scale controlled trials of antimicrobial-use regulation employing sophisticated epidemiologic methods, molecular typing, and precise resistance mechanism analysis.
Antimicrobial Stewardship(AMS) refers to the optimal selection, dosing, and duration of antimicrobial treatment resulting in the best clinical outcome with minimal side effects to the patients and minimal impact on subsequent resistance.
According to the 2019 report, in the US, more than 2.8 million antibiotic-resistant infections occur each year, and more than 35000 people die. In addition to this, it also mentioned that 223,900 cases of Clostridoides difficile occurred in 2017, of which 12800 people died. The report did not include viruses or parasites
VISION
Being proactive
Supporting optimal animal and human health
Exploring ways to reduce overall use of antimicrobials
Using the drugs that prevent and treat disease by killing microscopic organisms in a responsible way
GOAL
to prevent the generation and spread of antimicrobial resistance (AMR). Doing so will preserve the effectiveness of these drugs in animals and humans for years to come.
being to preserve human and animal health and the effectiveness of antimicrobial medications.
to implement a multidisciplinary approach in assembling a stewardship team to include an infectious disease physician, a clinical pharmacist with infectious diseases training, infection preventionist, and a close collaboration with the staff in the clinical microbiology laboratory
to prevent antimicrobial overuse, misuse and abuse.
to minimize the developme
The dimensions of healthcare quality refer to various attributes or aspects that define the standard of healthcare services. These dimensions are used to evaluate, measure, and improve the quality of care provided to patients. A comprehensive understanding of these dimensions ensures that healthcare systems can address various aspects of patient care effectively and holistically. Dimensions of Healthcare Quality and Performance of care include the following; Appropriateness, Availability, Competence, Continuity, Effectiveness, Efficiency, Efficacy, Prevention, Respect and Care, Safety as well as Timeliness.
2. Introduction
• Cell culture is the process by which prokaryotic,
eukaryotic or plant cells are grown under controlled
conditions. But in practice it refers to the culturing of
cells derived from animal cells.
• Cell culture was first successfully undertaken by Ross
Harrison in 1907
• Roux in 1885 for the first time maintained embryonic
chick cells in a cell culture
• Tool for the study of animal cell biology In vitro model of
cell growth
• Mimic of in vivo cell behaviour
• Artificial (some cell types are thus difficult to culture).
3. What is tissue culture?
• In vitro culture (maintain and/or
proliferate) of cells, tissues or organs
• Types of tissue culture
– Organ culture
– Tissue culture
– Cell culture
4. Organ culture
• The entire embryos or organs are excised from the
body and culture
• Advantages
– Normal physiological functions are maintained.
– Cells remain fully differentiated.
• Disadvantages
– Scale-up is not recommended.
– Growth is slow.
– Fresh explantation is required for every experiment.
5. Tissue Culture
• Fragments of excised tissue are grown in culture
media
• Advantages
– Some normal functions may be maintained.
– Better than organ culture for scale-up but not ideal.
• Disadvantages
– Original organization of tissue is lost.
6. Cell Culture
• Tissue from an explant is dispersed, mostly
enzymatically, into a cell suspension which may
then be cultured as a monolayer or suspension
culture.
• Advantages
– Development of a cell line over several generations
– Scale-up is possible
• Disadvantages
– Cells may lose some differentiated characteristics.
7. Why do we need Cell culture?
• Research
– To overcome problems in studying cellular behavior
such as:
• confounding effects of the surrounding tissues
• variations that might arise in animals under experimental
stress
– Reduce animal use
• Commercial or large-scale production
– Production of cell material: vaccine, antibody, hormone
8. Advantages
• Study of cell behaviour without the variations that
occur in animal
• Control of the growth environment leads to uniformity
of sample
• Characteristics of cells can be maintained over
several generations, leading to good reproducibility
between experiments
• Cultures can be exposed to reagents e.g. radio-
chemicals or drugs at defined concentrations
• Finally it avoids the legal, moral and ethical problems
of animal experimentation
9. Disadvantages
• Have to develop standardised techniques in
order to maintain healthy reproducible cells
for experiments
• Takes time to learn aseptic technique
• Quantity of material is limited
• Dedifferentiation and selection can occur and
many of the original cellular mechanisms can
be lost
10. Isolation of cell lines for in vitro culture
Resected Tissue
Cell or tissue culture in vitro
Primary culture
Secondary culture
Sub-culture
Cell Line
Sub-culture
Immortalization
Successive sub-culture
Single cell isolation
Clonal cell line Senescence
Transformed cell line
Immortalised cell line
Loss of control
of cell growth
11. 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.
12. Types of Cell culture
1. Primary Cultures
– Derived directly from excised tissue and cultured
either as
• Outgrowth of excised tissue in culture
• Dissociation into single cells (by enzymatic digestion or
mechanical dispersion)
– Advantages:
• usually retain many of the differentiated characteristics of
the cell in vivo
– Disadvantages:
• initially heterogeneous but later become dominated by
fibroblasts.
• the preparation of primary cultures is labor intensive
• can be maintained in vitro only for a limited period of time.
13. Types of Cell culture
2. Continuous Cultures
– derived from subculture (or passage, or transfer) of
primary culture
• Subculture = the process of dispersion and re-culture the
cells after they have increased to occupy all of the available
substrate in the culture
– usually comprised of a single cell type
– can be serially propagated in culture for several
passages
– There are two types of continuous cultures
• Cell lines
• Continuous cell lines
14. Types of continuous culture
1) Cell lines
• finite life, senesce after approximately thirty cycles of division
• usually diploid and maintain some degree of differentiation.
• it is essential to establish a system of Master and Working
banks in order to maintain such lines for long periods
2) Continuous cell lines
• can be propagated indefinitely
• generally have this ability because they have been
transformed
– tumor cells.
– viral oncogenes
– chemical treatments.
• the disadvantage of having retained very little of the original
in vivo characteristics.
15. Initiation of culture
Tissue
Primary cell culture
Cell line Continuous cell line
dispersion
Subculture
Finite numbers Indefinite numbers
Stored Stored
16. • Cell growth and differentiation in the
culture depends on:
– The nature of cells
– The culture environment
• the nature of the substrate on which cell grow
• the physicochemical and physiological
constitution of culture medium
• the constitution of gas phase
• the incubation temperature
• the cell-cell and cell-matrix interaction
17. Factors affecting cell behaviour in vivo
• The local micro-environment
• Cell-cell interactions
• Tissue architecture
• Tissue matrix
• Tissue metabolites
• Locally released growth factor and hormones
18. Culture Surface
• Most adherent cells require attachment to
proliferate
• Change charge of the surface
– Poly-L-lysine
• Coating with matrix proteins
– Collagen, laminin, gelatin, fibronectin
19. Media formulation
• Initial studies used body fluids
– Plasma, lymph, serum, tissue extracts
• Early basal media
– Salts, amino acids, sugars, vitamins supplemented
with serum
• More defined media
– Cell specific extremely complex
20. Media Formulation
• Inorganic ions
– Osmotic balance – cell volume
• Trace Elements
– Co-factors for biochemical pathways (Zn, Cu)
• Amino Acids
– Protein synthesis
– Glutamine required at high concentrations
• Vitamins
– Metabolic co-enzymes for cell replication
• Energy sources
– glucose
21. Serum provides the following
• Basic nutrients
• Hormones and growth factors
• Attachment and spreading factors
• Binding proteins (albumin, transferring)
carrying hormones, vitamins, minerals, lipids
• Protease inhibitors
• pH buffer
22. The gas phase
• Oxygen
– Aerobic metabolism
– Atmospheric 20%
– Tissue levels between 1-7%
• Carbon dioxide
– Buffering
23. pH Control
• Physiological pH 7
• pH can affect
– Cell metabolism
– Growth rate
– Protein synthesis
– Availability of nutrients
• CO2 acts as a buffering agent in combination
with sodium bicarbonate in the media
24. Temperature and Humidity
• Normal body temperature 37oC
• Humidity must be maintained at saturating
levels as evaporation can lead to changes in
– Osmolarity
– Volume of media and additives
25. Culture medium for animal cell
Appropriate medium
• Culture medium used need to :
i) meet basic nutritional requirement of
cells
ii) support growth of cells
iii) regulate the pH and osmolality
iv) provide essential gasses (O2 & CO2)
26. • Food portion of culture medium consist of :
a) Carbohydrates (glucose, fructose)
* provide an energy sources as well as a precursor for
biosynthesis
b) amino acids (Glutamine)
* as a sources of precursors for protein synthesis
Glutamine is normally included at higher concentrations in
order to act as a precursor for the TCA cycle intermediates.
However, ammonia is formed from the metabolic breakdown
of glutamine and can be inhibitory to growth in some cultures.
27. c) Vitamins & hormones
*are present at relatively low concentrations and are utilized
as metabolic cofactors.
Helps regulate and control the cell’s growth rate and functional
characteristics
d) Salts
*are included so that the solution is isotonic and has no
imbalances with the intracellular content
Helps regulate the flow of substances in and out of the cell
28. 5) Phenol red
*usually added as a pH indicator of the medium and
accounts for the color of culture media
6) Additional media supplements
• Serum – is a cell free-free liquid recovered from blood. Eg
fetal bovine serum, calf serum, horse serum
normally added to culture media to promote cell growth.
• Antibiotic – are often included in media for short-term
cultures in order to reduce the risk of contamination
29. Why sub culturing.?
• Once the available substrate surface is covered by
cells (a confluent culture) 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
30. 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 37 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
31. 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
32. 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 -80 C overnight
• Next day transfer the cryovials to Liquid nitrogen
33. Working with cryopreserved cells
• Vial from liquid nitrogen is placed into 37 C water bath,
agitate vial continuously until medium is thawed
• Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe top
of vial with 70% ethanol and discard the supernatant
• Resuspend the cell pellet in 1 ml of complete medium
with 20% FBS and transfer to properly labeled culture
plate containing the appropriate amount of medium
• Check the cultures after 24 hrs to ensure that they are
attached to the plate
• Change medium as the colour changes, use 20% FBS until
the cells are established
34. Contaminant’s of cell culture
Cell culture contaminants of two types
• Chemical-difficult to detect caused by endotoxins,
plasticizers, metal ions or traces of disinfectants
that are invisible
• Biological-cause visible effects on the culture they
are mycoplasma, yeast, bacteria or fungus or also
from cross-contamination of cells from other cell
lines
35. Contamination
Sources of Contamination of Biological-cause
• Bacteria
• Fungi
• Mould
• Yeast
• Mycoplasma
• Other cell types
• Free organisms, dust particles or aerosols
• Surfaces or equipment
36. Effects of Biological Contamination’s
• They competes for nutrients with host cells
• Secreted acidic or alkaline by-products ceses the
growth of the host cells
• Degraded arginine & purine inhibits the synthesis
of histone and nucleic acid
• They also produces H2O2 which is directly toxic to
cells
37. Detection of contaminants
• In general indicators of contamination are turbid culture
media, change in growth rates, abnormally high pH, poor
attachment, multi-nucleated cells, graining cellular appearance,
vacuolization, inclusion bodies and cell lysis
• Yeast, bacteria & fungi usually shows visible effect on the
culture (changes in medium turbidity or pH)
• Mycoplasma detected by direct DNA staining with intercalating
fluorescent substances e.g. Hoechst 33258
• Mycoplasma also detected by enzyme immunoassay by specific
antisera or monoclonal abs or by PCR amplification of
mycoplasmal RNA
• The best and the oldest way to eliminate contamination is to
discard the infected cell lines directly
38. Basic aseptic conditions
• If working on the bench use a Bunsen flame to heat the air
surrounding the Bunsen
• Swab all bottle tops & necks with 70% ethanol
• Flame all bottle necks & pipette by passing very quickly
through the hottest part of the flame
• Avoiding placing caps & pipettes down on the bench; practice
holding bottle tops with the little finger
• Work either left to right or vice versa, so that all material goes
to one side, once finished
• Clean up spills immediately & always leave the work place neat
& tidy
39. Safety aspect in cell culture
• Possibly keep cultures free of antibiotics in order to be able to
recognize the contamination
• Never use the same media bottle for different cell lines. If caps
are dropped or bottles touched unconditionally touched, replace
them with new ones
• Necks of glass bottles prefer heat at least for 60 secs at a
temperature of 200 C
• Switch on the laminar flow cabinet 20 mts prior to start working
• Cell cultures which are frequently used should be subcultered &
stored as duplicate strains
40. The Usage of Animal Cell Culture
1) Model System
Provide a good model system for studying
i) basic cell biology and biochemistry;
ii) interactions between disease-causing agents and
cells;
iii) effects of drugs on cells; process and triggers for
aging and nutritional studies.
41. 2) Toxicity Testing
Widely used to study the effects of new drugs,
cosmetics and chemicals on survival and growth in
wide variety of cell types.
3) Cancer Research
To study differences in both normal cells and cancer
cells.
To study the mechanism of cancer with the use of
use chemicals, viruses and radiation to convert
normal cultured cells to cancer causing cells.
42. 4) Virology
One of the earliest and major uses of cell culture is
the replication of viruses in cell cultures for use in
vaccine production.
Used in the clinical detection and isolation of viruses,
as well as basic research into how they grow and
infect organisms.
43. 5) Cell-Based Manufacturing
Three major areas cell-based industry are large-scale
production of :
i) viruses for use in vaccine production (polio, rabies, chicken
pox, hepatitis B and measles).
ii) cells that have been genetically engineered to produce
proteins that have medicinal or commercial value
(monoclonal antibodies, insulin, hormones).
iii) As replacement tissues and organs. Artificial skin for use
intreating burns and ulcers is the first commercially available
product.
A potentially supply of replacement cells and tissues may
come out of work currently being done with both embryonic
and adult stem cells.
44. 6) Genetic Counselling
Amniocentesis, a diagnostic technique that enables
doctors to remove and culture fetal cells from
pregnant women. These cells can be examined for
abnormalities in their chromosomes and genes.
7) Genetic Engineering
To reprogram cultured cells with new genetic
material (DNA and genes).
Also can be used to produce new proteins in large
quantity.
45. 8) Gene Therapy
The ability to genetically engineer cells has also led
to their use for gene therapy.
Cells can be removed from a patient lacking a
functional gene and the missing or damaged gene
can then be replaced.
9) Drug Screening and Development
Cell-based assays have become increasingly important
for the pharmaceutical industry as drugs.