Tissue Culture Intro 2011

8/2/2019 Tissue Culture Intro 2011

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BIOT 4620JA Negrn, Ph.D.

Introduction to Cell Tissue Culture

JA Negrn, Ph.D. BIOT 4620

Summary

Introduction to de course

Course syllabus

Introduction to tissue culture

Advantages, disadvantages, applications

Brief history

Media and growth requirements

Safety considerations


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IntroductionIntroductionIntroductionIntroduction Tissue Culture is a general term used for the

removal of cells, tissues, or organs from an

animal or plant and their subsequent placement

into an artificial environment conductive to

growth.

It consists of two types of culture:

Organ CultureOrgan Culture - Culture of whole organs or

intact organ fragments with the intent of

studying their continued function or

development

Cell CultureCell Culture - Culture of cells removed from theorgan fragments prior to or during cultivation


Tissue/cell culture Advantages

1. Controlled physiochemical environment (pH, temperature, osmotic

pressure, O2, CO2, etc.)

2. Controlled and defined physiological conditions (constitution of

medium, etc.)

3. Homogeneity of cell types (achieved through serial passages)

4. Economical, since smaller quantities of reagents are needed than in

vivo.

5. Legal moral and ethical questions of animal experimentation are

avoided.

Disadvantages1. Expertise is needed, so that behavior of cells in culture can be

interpreted and regulated.

2. Ten times more expensive for same quantity of animal tissue;

therefore reasons for its use should be compelling.

3. Unstable aneuploid chromosome constitution.


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Importance of Cell/Tissue Culture

Health

Pharmaceutical

Therapy

Agriculture

Food science

Breeding

Research

Team Generates 3-D Epithelial Tumors In

Vitro from Normal Human Primary Cells

GEN news highlights: Nov 22, 2010


Applications of Animal Tissue Culture

Model Systems

Toxicity testing

Cancer Research

Virology

Cell- Based Manufacturing

Genetic Counseling

Genetic Engineering

Gene Therapy Drug Screening and Development

Several therapies for human health

Therapy of burn victim patients


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Stem cells

Cells with the ability

to divide for indefinite

periods in culture and

to give rise to

specialized cells,

tissues, and organs.

Main types

Embryonic

Somatic

http://stemcells.nih.gov/info/basics/basics1.asp


Tissue Culture Brief History Tissue culture is often a generic

term that refers to both organ

culture and cell culture and the

terms are often used

interchangeably.

Cell cultures are derived from

either primary tissue explants or

cell suspensions.

Primary cell cultures typically

will have a finite life span inculture whereas continuous cell

lines are, by definition,

abnormal and are often

transformed cell lines.


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Tissue Culture Brief History

Ross Harrison

To grow his tissue explants, Harr ison adapted the

hanging drop technique that microbiologists used to

study live bacteria.

1907

Alexis Carrel

1910-1923

Adapted Harrison's hanging drop method to

work with warm blooded tissues by switching

from using clotted frog lymph to chicken

plasma clots to cultivate chick embryos.

"in recognition of his work on

vascular suture and the

transplantation of blood

vessels and organs"

Noble Prize 1912


1930s1930s1930s1930s Charles LindberghCharles LindberghCharles LindberghCharles Lindbergh AviatorAviatorAviatorAviator

and Cell and Organ Culturistand Cell and Organ Culturistand Cell and Organ Culturistand Cell and Organ Culturist

Developed a very efficient (>95%)centrifugal filter device forseparating serum from clottedplasma

Human serum was often preparedfrom blood obtained from a localhospital or from laboratoryworkers, while horses, rodents andchickens were the main sources of

animal blood. Serum needed to beprepared aseptically since it wasextremely difficult to filter throughthe porcelain, asbestos or sinteredglass filters available in the 1930s. Lindbergh centrifugal filter tubes

designed for efficiently extracting

serum from clotted plasma


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Blood plasma vs Serum

Plasma, is prepared by obtaining a sample

of blood and removing the blood cells by

spinning with a centrifuge.

Serum is prepared by obtaining a blood

sample, allowing formation of the blood

clot, and removing the clot using a

centrifuge

Blood plasma vs Serum

Protein type % inserum

% inblood

g/lblood

Primary function/s

Total 60-84

Albumin 53% 61 35-50capillary colloid osmotic pressure ->reduces fluid leakage out of capillariesTransport

Globulinalpha

beta

gamma

14%

12%

20%

34 23-35 Transport, substrates for formation ofother substances

Transport, substrates for formation ofother substancesimmunity [antibodies]

Fibrinogen NA 4.11.9-3.6

Blood clotting


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1950s1950s1950s1950s ---- Spinner Flasks to BioreactorsSpinner Flasks to BioreactorsSpinner Flasks to BioreactorsSpinner Flasks to Bioreactors

1953

The first successful suspension cultures, lymphoblastic MBIII cells, were

grown by Owens.

1954

Earle and his group extended these studies to L929 mouse cells growing

them in roller tubes (10+ RPM) to prove that normally attached cells

could grow in suspension.

Erlenmeyer flasks on rotary shakers

1956

Cherry and Hull used a magnetic stir bar suspended from a fishing

swivel to keep cells in suspension in a round bottom flask so that they

could study the effects of varying the medium, speed and seeding

densities on cell growth.

1957 McLimans and his group (6) took the magnetic stir bar and attached it

to a sliding wire so that it could be raised a lowered to match the

medium volume. They added a side port for sampling and called it a

spinner flask

McLimans' "spinner flasks

Erlenmeyer flasks


Modern Fermentors and Bioreactors Fermentation is an energy-yielding

anaerobic metabolic process in

which organisms convert nutrients

(typically carbohydrates) to

alcohols and acids (lactic acid and

acetic acid).

Conversion of sugar to alcohol, using

yeast, under anaerobic conditions, as in

the production of beer or wine,

vinegars and cider.

In biotechnology, the term is used more

loosely to refer to growth of

microorganisms on food, under eitheraerobic or anaerobic conditions.

Bioreactors degrade contaminants

in water with microorganisms.

by Genentech


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Tissue Culture MethodsTissue Culture MethodsTissue Culture MethodsTissue Culture Methods

WORK AREA AND EQUIPMENT Laminar flow hoods CO2 Incubators Microscopes

Inverted phase Contrast microscopes

Preservation Liquid N2

Vessels

MAINTENANCE Growth pattern Harvesting

Suspension culture Adherent cultures


Media and growth requirementsMedia and growth requirementsMedia and growth requirementsMedia and growth requirements

Media and growth requirements

Physiological parameters

A. temperature - 37C for cells from homeother

B. pH - 7.2-7.5 and osmolality of medium must be maintained

C. humidity is required

D. gas phase - bicarbonate conc. and CO2 tension in

equilibrium

E. visible light - can have an adverse effect on cells; lightinduced production of toxic compounds can occur in some

media; cells should be cultured in the dark and exposed to

room light as little as possible


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Medium Requirements

Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2 Trace elements - iron, zinc, selenium

sugars - glucose is the most common

amino acids - 13 essential

vitamins - B, etc.F. choline, inositol G. serum - contains a large

number of growth promoting activities such as buffering toxic

nutrients by binding them, neutralizes trypsin and other

proteases, has undefined effects on the interaction between cells

and substrate, and contains peptide hormones or hormone-like

growth factors that promote healthy growth.H. antibiotics - although not required for cell growth,

antibiotics are often used to control the growth of bacterial and

fungal contaminants.


Measurement of growth and viability The viability of cells can be observed

visually using an inverted phasecontrast microscope.

Live cells are phase bright;suspension cells are typicallyrounded and somewhat symmetrical;adherent cells will form projectionswhen they attach to the growthsurface.

Viability can also be assessed usingthe vital dye, trypan blue, which is

excluded by live cells butaccumulates in dead cells. Cellnumbers are determined using ahemocytometer.


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SAFETY CONSIDERATIONSSAFETY CONSIDERATIONSSAFETY CONSIDERATIONSSAFETY CONSIDERATIONS Contaminating pathogenic agents

Contamination with adventitious agents such as bacteria, fungi, mycoplasma,and virus

International Classification Microorganisms in Risk Group 1

Are not identified as causative agents of disease in humans and that are not a treat forthe environment.

Microorganisms in Risk Group 2 May cause disease in humans and therefore offer a hazard to the lab workers. Are

unlikely to spread into the environment. Prophylactics available.

Microorganisms in Risk Group 3 Offer severe treat to the health of lab workers but small risk to the population.

Prophylactics available.

Microorganisms in Risk Group 4 Cause severe illness in humans and offer a serious hazard to the lab workers and to the

population. Prophylactics not available.

http://www.biosafety.be/RA/Class/ClassINT.html


SAFETY CONSIDERATIONSSAFETY CONSIDERATIONSSAFETY CONSIDERATIONSSAFETY CONSIDERATIONS National Institutes of Health (NIH) - Classification of human etiologic

agents on the basis of hazard (2002)

Risk Group 1 (RG1) Agents that are not associated with disease in healthy adult humans.

Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis, Escherichia

coli-K12, and adeno-associated virus (AAV) types 1 through 4.

Risk Group 2 (RG2) Agents that are associated with human disease which is rarely serious and

for which preventive or therapeutic interventions are often available.

Examples of RG2 agents include Salmonella sp., Chlamydia psittaci, measles virus, and hepatitis A,

B, C, D, and E viruses.

Risk Group 3 (RG3) Agents that are associated with serious or lethal human disease for which

preventive or therapeutic interventions may be available (high individual risk but low community

risk).

Examples of RG3 agents include Brucella, Mycobacterium tuberculosis, Coccidioides immitis, yellow

fever virus and human immunodeficiency virus (HIV) types 1 and 2.

Risk Group 4 (RG4) agents are likely to cause serious or lethal human disease for which

preventive or therapeutic interventions are not usuallyavailable (high individual risk and high

community risk). RG4 agents only include viruses.

Examples of RG4 agents include Crimean-Congo hemorrhagic fever virus, Ebola virus and

herpesvirus simiae (B-virus).

http://oba.od.nih.gov/rdna/nih_guidelines_oba.html


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Risk when handling animal cell culture Microorganisms with higher potential to cause human contamination

Viruses May produce no cytopathic effect

May be latent

Difficult to detect

May come from donor or from contamination by the operator during the cell culture process (enzymes,serum, proteins, fetal extracts, hormones, growth factors, other)

Bacteria and fungi In general can be detected in cell culture (cell death, pH, turbidity)

Mycoplasma Special hazardous in cell culture products

Tolerant to common antibiotics

Careful and routine testing is necessary

Parasites A concern in freshly prepared primary cell culture or organ culture

Prions

Infectious agent composed of protein in a misfolded form Neurodegerative diseases in animals and humans


Cell culture contamination Bacteria

Bacterial contamination is usually manifest

by a sudden change in pH

Fungi

Fungi or molds produce thin filamentous

mycelia and sometimes denser clumps of

spores

Mycoplasma

Cannot be detected by typical light

microscopy

100X

10X, 40X Fungi

Special

culture

method


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SAFETY CONSIDERATIONSSAFETY CONSIDERATIONSSAFETY CONSIDERATIONSSAFETY CONSIDERATIONS Assume all cultures are hazardous since they may harbor latent viruses

or other organisms that are uncharacterized. The following safetyprecautions should also be observed:

pipetting: use pipette aids to prevent ingestion and keep aerosols down to a

minimum

no eating, drinking, or smoking

wash hands after handling cultures and before leaving the lab

decontaminate work surfaces with disinfectant (before and after)

autoclave all waste

use biological safety cabinet (laminar flow hood) when working with

hazardous organisms. The cabinet protects worker by preventing airborne cells

and viruses released during experimental activity from escaping the cabinet; there

is an air barr ier at the front opening and exhaust air is filtered with a HEPA filter

make sure cabinet is not overloaded and leave exhaust grills in the front and theback clear (helps to maintain a uniform airflow)

use aseptic technique

dispose of all liquid waste after each experiment and treat with bleach


99999999--------10 Days Chick10 Days Chick10 Days Chick10 Days Chick10 Days Chick10 Days Chick10 Days Chick10 Days Chick--------EmbryoEmbryoEmbryoEmbryoEmbryoEmbryoEmbryoEmbryo

Air sac

Yolk sac

Albumin

Chorioallantoic

membrane

Allantoic

cavity

http://www.science-art.com/image/?id=2051


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Culturing Primary Chick Embryo Cells

Materials

Fertile Eggs (10 to 12 day gestation)

Minimum Essential Medium Eagle

(MEM) or HanksBalanced Salt Solution

Bovine Calf Serum (BCS), iron

supplemented or Newborn Calf Serum

(NCS)

L-Glutamine, 200 mM

Non-essential Amino Acids (NEAA),

100X

Dulbeccos Phosphate Buffered Saline,

1X (without Ca2+ or Mg2+)

Trypsin Solution, 1X Antibiotic/Antimycotic Solution, 100X

Crystal Violet or Methyl Violet, 0.1 -

0.4 % in aqueous alcohol solution

Materials RPMI-1640 Medium

8-11 days fertile eggs

Sterile 150 x 15 mm Petri dishes

70% ethanol

Sterile scissors

Sterile forceps

Sterile spatulas

1X PBS

250mL sterile beakers

Sterile gauze

50mL conical sterile centrifuge tubes

37C water bath

Trypan Blue

crystal violet or methyl violet stain0.1 - 0.4%

Solutions Prepare growth medium for

primary chick embryo cells asfollows: HanksBalanced Salt Solution,1X

45.0 mL BCS 5.0 mL (bovine calf serum or

fetal calf serum)

PBS (Phosphate BufferSolution) 137 mM NaCl 2.7 mM KCl 10 mM Na2PO4 1.76 mM KH2PO4 pH 7.4

Dissolve in 800ml distilledH2O.

Adjust pH to 7.4.

Adjust volume to 1L withadditional distilled H2O.

Sterilize by autoclaving


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Homework

Lindee, S.M. 2007. The Culture of Cell Culture. Science.

316(5831): 1568-1569.

Introduction to Animal Cell Culture. Corning Incorporated.

http://www.level.com.tw/html/ezcatfiles/vipweb20/img/

img/20297/intro_animal_cell_culture.pdf

Tissue Culture Intro 2011

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