Animal Cell1

7/31/2019 Animal Cell1

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Animal cell culture Cell culture cells are grown under controlled conditions

Cells can grow in simple glass or plastic containers with essential requirements for

animal cell culture (levels of oxygen, carbon dioxide, temperature, humidity as

present in the animals body).

There is a difference in the in vitro and in vivo growth pattern of cells For example

(i) there is an absence of cell-cell interaction and cell matrix interaction,

(ii) there is a lack of three-dimensional architectural appearance

(iii) changed hormonal and nutritional environment.


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Basic equipments used in cell culture

Laminar cabinet-Vertical are preferable

Incubation facilities- Temperature of 25-30 C for insect & 37 C for

mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To prevent

cell death incubators set to cut out at approx. 38.5 C

Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media

components (e.g. glutamine & serum) at -20 C

Microscope- An inverted microscope with 10x to 100x magnification

Tissue culture ware- Culture plastic ware treated by polystyrene


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Mechanics of phase microscopy

Shifting of phase by a wavelength

Add and subtract amplitudes to create

more contrast


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Anchorageindependant cells

Cells associated with body fluid blood cells

Grown in suspension

Will eventually need subculturing

Anchoragedependant cells

Most animal derived cells

Adhere to bottom of a flask and form a monolayer

Eventually cover entire surface of substratum (confluence)

Proliferation then stops

Need to subculture cells at this point (remove to fresh medium)

Proliferation can begin again


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Basic Cell Culture Procedure for Anchorage Dependent

Cells

View cells using inverted phase microscope

Aseptically aspirate media

Rinse media with PBS

Add Trypsin-EDTA to cells

Aspirate Trypsin-EDTA

Incubate cells with layer of Trypsin-EDTA at 37 C

Resuspend cells with fresh media

Take sample and count cells

Calculate how many cells are needed to add to new plate or flask


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Classification of Cell Cultures

Primary cell cultures

Cells that are cultured directly from a Animal.

Most have climited lifespan. (After a certain number of population doublings cells undergo the process of

senescence and stop dividing, while generally retaining viability.)

Secondary Culture

Cells taken from a primary culture and passed or divided in vitro.

These cells have a limited number of divisions or passages. After the limit, they will undergo apoptosis.
http://en.wikipedia.org/w/index.php?title=Primary_cell_culture&action=edit&redlink=1http://en.wikipedia.org/wiki/Senescencehttp://en.wikipedia.org/wiki/Senescencehttp://en.wikipedia.org/w/index.php?title=Primary_cell_culture&action=edit&redlink=1


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Primary culture

Cells when surgically or enzymatically removed from an organism

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

Cells such as macrophages and neurons do not divide in vitro so can be used as primary

cultures

Lineage of cells originating from the primary culture is called a cell strain


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Desinfection step

Tissue isolation

Incubation&growth

Primary cells


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Making a Primary Culture


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Continous cell lines

Cell lines which either occur spontaneously or induced virally or chemically transformed into

Continous cell lines

Characteristics of continous cell lines

Smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio

Ability to grow upto higher cell density

After a limited number of passages they will grow indefinitely.

Two cell types fused together with characteristics of each


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Common cell lines

Human cell lines

-MCF-7 breast cancer

HL 60 Leukemia

HEK-293 Human embryonic kidney

HeLa Henrietta lacks

Primate cell lines

Vero African green monkey kidney epithelial cells

Cos-7 African green monkey kidney cells

And others such as CHO from hamster, sf9 & sf21 from insect cells


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Serum-free media are media designed to grow a specific cell type or perform a specific

application in the absence of serum.

Advantages of using serum-free media:

Increased definition.

More consistent performance.

Easier purification and downstream processing.

Precise evaluations of cellular function.

Increased growth and/or productivity.

Better control(s) over physiological responsiveness.

Enhanced detection of cellular mediators.


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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


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PRESERVATION AND STORAGE.

Freezing can be lethal to cells due to the effects of damage by

Ice crystals,Alterations in the concentration of electrolytes,

Dehydration,

Changes in pH.

To minimize the effects of freezing,

First, a cryoprotective agent which lowers the freezing point, (glycerol or

DMSO, is added). Atypical freezing medium is 90% serum, 10% DMSO. In

addition,

use healthy cells that are growing in log phase (replace the medium 24 hours

before freezing).


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Two methods of freezing

Snap freezing

Liquid N2 is used to preserve tissue culture cells, (liquid phase (-196C) or in the vapor

phase (-156C)).

Deep freezing

Also, the cells are slowly cooled from room temperature to -80C to allow the water to

move out of the cells before it freezes.

The optimal rate of cooling is 1-3C per minute.


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Working with cryopreserved cells

To maximize recovery of the cells when thawing, the cells are warmed very

quickly by placing the tube directly from the liquid nitrogen container into a

37C water bath with moderate shaking.

As soon as the last ice crystal is melted, the cells are immediately diluted into

prewarmed medium.

Centrifuge the vial for 10 mts at 1000 rpm at RT, and add fresh media.

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


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Cell viability

Cell viability is determined by staining the cells with trypan blue

As trypan blue dye is permeable to non-viable cells or death 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= Nu. of unstained cells x 100

total nu. of cells

C lt res sho ld be e amined dail obser ing the morpholog the color of the medi m and the


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Cultures should be examined daily, observing the morphology, the color of the medium and the

density of the cells.

Growth pattern.

Cells will initially go through a quiescent or lag phase that depends on the cell type, the

seeding density, the media components, and previous handling.

The cells will then go into exponential growth where they have the highest metabolic activity.

The cells will then enter into stationary phase where the number of cells is constant, this is

characteristic of a confluent population (where all growth surfaces are covered).Harvesting.

Cells are harvested when the cells have reached a population density which suppresses growth.

Ideally, cells are harvested when they are in a semi-confluent state and are still in log phase. Cells

that are not passaged and are allowed to grow to a confluent state can sometime lag for a long

period of time and some may never recover.

Most cells are passaged (or at least fed) three times a week.

Growth and morphology


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Growth and morphology

Visually inspect cells frequently. Cell culture is sometimes more an art than a science.

Frequent feeding is important for maintaining the pH balance of the medium and for eliminating

waste products.

Cells do not typically like to be too confluent so they should be subcultured when they are in a

semi-confluent state.

In general, mammalian cells should be handled gently. They should not be vortexed, vigorously

pipetted or centrifuged at greater than 1500 g.Cell feeding.

Use prewarmed media and have cells out of the incubator for as little time as possible.

Use 10-15 ml for T-25's, 25-35 ml for T-75's and 50-60 ml for T-150's. a. Suspension cultures.

Feeding and subculturing suspension cultures are done simultaneously.

The dilution you use will depend on the density of the cells and how quickly they divide, which

only you can determine.

Typically 1:4 to 1:20 dilutions are appropriate for most cell lines.


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Animal Cell1